A pharmaceutical composition containing an antibody-drug conjugate and its use

Abstract

Provided is a pharmaceutical composition containing an antibody-drug conjugate and its use. Specifically provided is a pharmaceutical composition comprising an anti-claudin antibody-drug conjugate.

Description

[0001] This application claims the priority of Chinese Patent Application No. 202011061863.1 filed on September 30, 2020 and Chinese Patent Application No. CN202111069020.0 filed on September 13, 2021. Technical Field

[0002] This disclosure belongs to the field of pharmaceutical preparations, and particularly relates to a pharmaceutical composition comprising an antibody-drug conjugate and its use as an anti-cancer drug. Background Art

[0003] The statements herein only provide background information related to this disclosure and do not necessarily constitute prior art.

[0004] Claudin-18 is a protein encoded by the Claudin18 gene in humans and belongs to the family of cell tight junction proteins, which can control the molecular flow between epithelial cells. The Claudin protein structure includes four transmembrane regions and two extracellular loops (with their N-terminus and C-terminus in the cytoplasm).

[0005] Claudin-18 has two splicing variants, namely Claudin 18.1 and Claudin 18.2, and there are only eight amino acid differences between their sequences in the first extracellular loop. The expression distributions of Claudin 18.1 and Claudin 18.2 are different. Claudin 18.1 is selectively expressed in normal lung cells, while the expression of Claudin 18.2 is highly restricted in normal cells but is frequently ectopically activated and overexpressed in various tumors (such as gastric cancer, lung cancer, and pancreatic cancer, etc.). Claudin18.2 is considered a potential therapeutic target for gastric cancer and other cancer types, and the discovery of this target also provides a new option for the treatment of gastric cancer.

[0006] An antibody-drug conjugate (ADC) links a monoclonal antibody or antibody fragment to a bioactive cytotoxin through a stable chemical linker compound, making full use of the specificity of the antibody for binding to surface antigens of normal cells and tumor cells and the high efficiency of the cytotoxic substance, while avoiding the defects of low efficacy of the former and excessive toxic side effects of the latter. This means that, compared with traditional chemotherapy drugs in the past, antibody-drug conjugates can bind to tumor cells more precisely and reduce the impact on normal cells.

[0007] Currently, there have been patent reports on antibodies and ADC drugs targeting Claudin18.2, such as WO2016166122 and WO2016165762.

[0008] Because ADCs have a more complex heterogeneous structure than antibodies, greater challenges are posed to ADC preparations for therapeutic purposes. SUMMARY OF THE INVENTION

[0009] The present disclosure relates to pharmaceutical preparations containing anti-Claudin18.2 antibody-drug conjugates and their uses. The preparations have advantages such as good stability and good lyophilized form.

[0010] The present disclosure provides a pharmaceutical composition comprising an anti-Claudin18.2 antibody-drug conjugate and a buffer, wherein the anti-Claudin18.2 antibody in the anti-Claudin18.2 antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein:

[0011] i) The heavy chain variable region has HCDR1, HCDR2, and HCDR3 with the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 6; or

[0012] ii) The heavy chain variable region has HCDR1, HCDR2, and HCDR3 with the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 3, and the light chain variable region has LCDR1, LCDR2, and LCDR3 with the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 4;

[0013] The buffer is a histidine salt buffer.

[0014] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the buffer is a histidine-acetate buffer.

[0015] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0016] iii) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 respectively; or

[0017] (iv) The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.

[0018] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the anti-Claudin18.2 antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0019] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0020] (1) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto;

[0021] (2) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 24 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 21 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto;

[0022] (3) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 5 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 6 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; or

[0023] (4) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 31 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 28 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0024] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the anti-Claudin18.2 antibody is a humanized antibody, and the humanized antibody comprises a framework region derived from a human antibody or a variant thereof, and the framework region variant has at most 10 amino acid back mutations on the light chain framework region and / or the heavy chain framework region of the human antibody.

[0025] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the framework region variant comprises a mutation selected from the following (a) or (b):

[0026] (a) The light chain variable region comprises one or more amino acid back mutations selected from any of 22S, 85I and 87H, and / or the heavy chain variable region comprises one or more amino acid back mutations selected from any of 48I, 82T and 69M; or

[0027] (b) The light chain variable region comprises one or more amino acid back mutations selected from any of 4L or 22S, and / or the heavy chain variable region comprises one or more amino acid back mutations selected from any of 38K, 40R, 48I, 66K, 67A, 69L, 71L and 73K.

[0028] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the framework region variant comprises a mutation selected from the following:

[0029] (a-1) The light chain variable region comprises amino acid back mutations of 22S, 85I and 87H, and the heavy chain variable region comprises amino acid back mutations of 48I and 82T; or

[0030] (b-1) The light chain variable region comprises an amino acid back mutation of 4L;

[0031] Wherein, 82 in 82T of the heavy chain variable region is the 82A position according to Kabat rules.

[0032] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region as shown in any of the following:

[0033] (vii) The heavy chain variable region is as shown in SEQ ID NO: 3 and the light chain variable region is as shown in SEQ ID NO: 4;

[0034] (viii) The heavy chain variable region is as shown in SEQ ID NO: 24, 25, 26 or 27 and the light chain variable region is as shown in SEQ ID NO: 21, 22 or 23;

[0035] (ix) The heavy chain variable region is as shown in SEQ ID NO: 5 and the light chain variable region is as shown in SEQ ID NO: 6; or

[0036] (x) The heavy chain variable region is as shown in SEQ ID NO: 31, 32, 33 or 34 and the light chain variable region is as shown in SEQ ID NO: 28, 29 or 30;

[0037] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region as shown in any of the following:

[0038] (xi) The heavy chain variable region is as shown in SEQ ID NO: 31 and the light chain variable region is as shown in SEQ ID NO: 29; or

[0039] (xii) The heavy chain variable region is as shown in SEQ ID NO: 26 and the light chain variable region is as shown in SEQ ID NO: 23.

[0040] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the anti-Claudin18.2 antibody comprises an antibody heavy chain constant region and a light chain constant region.

[0041] In some embodiments, the heavy chain constant region is selected from human IgG1, IgG2, IgG3 and IgG4 constant regions and their conventional variants, and the light chain constant region is selected from human antibody κ and λ chain constant regions and their conventional variants. In some embodiments, the antibody comprises a heavy chain constant region as shown in SEQ ID NO: 7 and a light chain constant region as shown in SEQ ID NO: 8.

[0042] In some embodiments, the antibody comprises: a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the heavy chain having the amino acid sequence shown in SEQ ID NO: 35 or 42, and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the light chain having the amino acid sequence shown in SEQ ID NO: 36 or 39; or a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the heavy chain having the amino acid sequence shown in SEQ ID NO: 37 or 49, and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the light chain having the amino acid sequence shown in SEQ ID NO: 38 or 46.

[0043] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the anti-Claudin18.2 antibody comprises a heavy chain and a light chain as shown in any of the following:

[0044] (c) a heavy chain with the sequence as shown in SEQ ID NO: 35 and a light chain with the sequence as shown in SEQ ID NO: 36;

[0045] (d) a heavy chain with the sequence as shown in SEQ ID NO: 42, 43, 44 or 45 and a light chain with the sequence as shown in SEQ ID NO: 39, 40 or 41;

[0046] (e) a heavy chain with the sequence as shown in SEQ ID NO: 37 and a light chain with the sequence as shown in SEQ ID NO: 38; or

[0047] (f) a heavy chain with the sequence as shown in SEQ ID NO: 49, 50, 51 or 52 and a light chain with the sequence as shown in SEQ ID NO: 46, 47 or 48.

[0048] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the anti-Claudin18.2 antibody comprises a heavy chain and a light chain as shown in any of the following:

[0049] a heavy chain as shown in SEQ ID NO: 44, and a light chain as shown in SEQ ID NO: 41; or

[0050] a heavy chain as shown in SEQ ID NO: 49, and a light chain as shown in SEQ ID NO: 47.

[0051] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the anti-Claudin18.2 antibody-drug conjugate has a structure represented by the general formula (Pc-L-Y-D):

[0052]

[0053] Wherein:

[0054] Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-;

[0055] R a and R b are the same or different and each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclic group;

[0056] Alternatively, R a and R b together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclic group;

[0057] R 1 is selected from a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;

[0058] R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;

[0059] Alternatively, R 1 and R 2 together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclic group;

[0060] Alternatively, R a and R 2 together with the carbon atom to which it is attached form a cycloalkyl or heterocyclic group;

[0061] m is an integer from 0 to 4;

[0062] n is from 1 to 10, and n is a fraction or an integer;

[0063] L is a linker unit;

[0064] Pc is an anti-Claudin18.2 antibody;

[0065] In some embodiments, for the pharmaceutical composition as described in any one of the above, wherein n is a fraction or an integer, it can be 2 to 8, 3 to 7, 3.5 to 4.5, 2, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9.

[0066] In some embodiments, for the pharmaceutical composition as described in any one of the above, wherein the anti-Claudin18.2 antibody-drug conjugate has a structure represented by the general formula (Pc-L-Y-D),

[0067] wherein:

[0068] Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-;

[0069] R a and R b are the same or different and each independently selected from a hydrogen atom, a deuterium atom, a halogen or a C 1-6 alkyl;

[0070] R 1 is a halo C 1-6 alkyl or a C 3-6 cycloalkyl;

[0071] R 2 is selected from a hydrogen atom, a halo C 1-6 alkyl or a C 3-6 cycloalkyl;

[0072] Alternatively, R 1 and R 2 together with the carbon atom to which it is attached form a C 3-6 cycloalkyl;

[0073] m is 0 or 1.

[0074] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the anti-Claudin18.2 antibody-drug conjugate has a structure represented by the general formula (Pc-L-Y-D), wherein Y is selected from:

[0075]

[0076] wherein the O-terminus of Y is linked to the linker unit L.

[0077] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0078] L 1 is selected from -(succinimidyl-3-yl-N)-W-C(O)-, -CH 2 -C(O)-NR 3 -W-C(O)- or -C(O)-W-C(O)-, wherein W is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl or a straight-chain heteroalkyl of 1 to 8 chain atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 alkyl, C 1-8 alkyl-cycloalkyl or a straight-chain heteroalkyl of 1 to 8 chain atoms is each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, C 1-6 alkyl, chloro-C 1-6 alkyl, deuterated-C 1-6 alkyl, C 1-6 alkoxy and C 3-6 cycloalkyl;

[0079] L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 )p 1 C(O)- or a chemical bond, wherein p 1 is an integer from 1 to 20;

[0080] L 3The peptide residue is composed of 2 to 7 amino acids, wherein the amino acid residue is selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and is optionally further selected from halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, Chloro C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 The cycloalkyl group is substituted by one or more substituents;

[0081] L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 -、-C(O)NR 5 (CH 2 ) t - or a chemical bond, wherein t is an integer from 1 to 6;

[0082] R 3 , R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, a C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl and C 1-6 Hydroxyalkyl;

[0083] R 6 and R 7 are the same or different and are each independently selected from hydrogen atoms, halogens, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl and C 1-6 Hydroxyalkyl.

[0084] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0085] L 1 for s 1 is an integer from 2 to 8;

[0086] L 2 is a chemical bond;

[0087] L 3is a tetrapeptide residue; preferably a tetrapeptide residue of GGFG (SEQ ID No: 55);

[0088] L 4 is -NR 5 (CR 6 R 7 )t-, R 5 、R 6 or R 7 are the same or different and each independently is a hydrogen atom or a C 1-6 alkyl group, and t is 1 or 2;

[0089] wherein the L 1 terminus is connected to Pc, and the L 4 terminus is connected to Y.

[0090] In some embodiments, for the pharmaceutical composition as described in any one of the above, wherein -L- is:

[0091]

[0092] In some embodiments, for the pharmaceutical composition as described in any one of the above, wherein L-Y- is optionally selected from:

[0093]

[0094] In some embodiments, for the pharmaceutical composition as described in any one of the above, wherein the anti-Claudin18.2 antibody-drug conjugate is selected from the structures shown in any of the following:

[0095]

[0096]

[0097] wherein Pc and n are as defined in the general formula (Pc-L-Y-D).

[0098] In some embodiments, for the pharmaceutical composition as described in any one of the above, wherein the anti-Claudin18.2 antibody-drug conjugate has the structure shown in the following formula:

[0099]

[0100] wherein:

[0101] n is from 2 to 8, and n is a decimal or an integer;

[0102] Pc is an anti-Claudin18.2 antibody.

[0103] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the pharmaceutical composition further comprises a surfactant. In some embodiments, the surfactant is selected from polysorbates (such as polysorbate 20, polysorbate 80), polyhydroxyalkenes, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauroyl sarcosine, myristoyl sarcosine, linoleoyl sarcosine, stearoyl sarcosine, linoleoyl betaine, myristoyl betaine, cetyl betaine, lauramidopropyl betaine, cocoamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium methyl cocoyl taurate, sodium methyl oleoyl taurate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, and the like.

[0104] In some embodiments, the surfactant is a polysorbate. In some embodiments, the surfactant is polysorbate 80 or polysorbate 20. In some embodiments, the surfactant is polysorbate 80.

[0105] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the surfactant is 0.05 mg / mL to 0.5 mg / mL or 0.1 mg / mL to 0.2 mg / mL. In some embodiments, the concentration of the surfactant is 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.18 mg / mL, 0.19 mg / mL, 0.2 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL. In some embodiments, the concentration of the surfactant is 0.2 mg / mL.

[0106] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the composition further comprises a sugar. In some embodiments, the sugar is selected from conventional compositions (CH 2 O) nand its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. The sugar described above can be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerin, arabitol, sylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, manninotriose, stachyose, maltose, lactulose, palatinose, sorbitol, maltitol, lactitol, isomaltulose, etc. In some embodiments, the sugar is selected from sucrose, mannitol, and trehalose. In some embodiments, the sugar is sucrose.

[0107] In some embodiments, for the pharmaceutical composition described in any one of the above, the sugar concentration is 20 mg / mL to 100 mg / mL or 40 mg / mL to 80 mg / mL. In some embodiments, the sugar concentration is 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL. In some embodiments, the sugar concentration is 40 mg / mL.

[0108] In some embodiments, for the pharmaceutical composition described in any one of the above, the antibody-drug conjugate concentration is 1 mg / mL to 100 mg / mL based on the protein (i.e., antibody) concentration or 10 mg / mL to 30 mg / mL based on the protein concentration. In some embodiments, the antibody-drug conjugate concentration is 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL based on the protein concentration. In some embodiments, the antibody-drug conjugate concentration is 20 mg / mL based on the protein concentration.

[0109] In some embodiments, for the pharmaceutical composition described in any one of the above, the concentration of the buffer is 5 mM to 50 mM or 10 mM to 30 mM. In some embodiments, the concentration of the buffer is 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. In some embodiments, the concentration of the buffer is 30 mM.

[0110] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the pH of the pharmaceutical composition is 5.0 - 6.5 or 5.0 - 5.5. In some embodiments, the pH of the pharmaceutical composition is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5. In some embodiments, the pH of the pharmaceutical composition is 5.0 - 5.4. In some embodiments, the pH of the pharmaceutical composition is 5.0 - 5.3.

[0111] In some embodiments, the pharmaceutical composition as described in any one of the above, which comprises the following components:

[0112] (a) The anti-Claudin18.2 antibody-drug conjugate at a protein concentration of 10 mg / mL to 30 mg / mL, (b) polysorbate at 0.1 mg / mL to 0.2 mg / mL, (c) sugar at 40 mg / mL to 80 mg / mL, and (d) histidine salt buffer at 10 mM to 30 mM; the pH of the pharmaceutical composition is about 5.0 - 5.5.

[0113] In some embodiments, the pharmaceutical composition as described in any one of the above, which comprises the following components:

[0114] (a) The anti-Claudin18.2 antibody-drug conjugate at a protein concentration of 10 mg / mL to 30 mg / mL, (b) polysorbate 80 at 0.1 mg / mL to 0.2 mg / mL, (c) sucrose at 40 mg / mL to 80 mg / mL, and (d) histidine salt buffer at 10 mM to 30 mM; the pH of the pharmaceutical composition is about 5.0 - 5.5.

[0115] In some embodiments, the pharmaceutical composition as described in any one of the above, which comprises the following components:

[0116] (a) The anti-Claudin18.2 antibody-drug conjugate at a protein concentration of 20 mg / mL, (b) polysorbate 80 at 0.2 mg / mL, (c) sucrose at 40 mg / mL, and (d) 30 mM histidine-acetate buffer, the pH of the pharmaceutical composition is 5.0 - 5.3.

[0117] In some embodiments, for the pharmaceutical composition as described in any one of the above, the anti-Claudin18.2 antibody-drug conjugate has the structure shown in the following formula:

[0118]

[0119] Wherein:

[0120] n is from 2 to 8, and n is a decimal or an integer;

[0121] Pc is an anti-Claudin18.2 antibody, which comprises a heavy chain shown in SEQ ID NO: 49 and a light chain shown in SEQ ID NO: 47; the pharmaceutical composition contains the antibody-drug conjugate at a protein concentration of 20 mg / mL;

[0122] The pharmaceutical composition further comprises the following components:

[0123] 0.2 mg / mL of polysorbate 80, 40 mg / mL of sucrose and 30 mM of histidine-acetate buffer, and the pH of the pharmaceutical composition is 5.0 - 5.3.

[0124] In some embodiments, for the pharmaceutical composition as described in any one of the above, the pharmaceutical composition is a liquid preparation. In some embodiments, the solvent of the liquid preparation is water.

[0125] The present disclosure also provides a lyophilized preparation containing an antibody-drug conjugate, which is characterized in that the preparation can form the pharmaceutical composition as described in any one of the above after reconstitution.

[0126] The present disclosure also provides a lyophilized preparation, which is the lyophilized form of the pharmaceutical composition as described in any one of the above.

[0127] The present disclosure also provides a method for preparing a lyophilized preparation containing an antibody-drug conjugate, which includes the step of freeze-drying the pharmaceutical composition as described in any one of the above.

[0128] The present disclosure also provides a lyophilized preparation containing an antibody-drug conjugate, which is obtained by freeze-drying the pharmaceutical composition as described in any one of the above.

[0129] In some embodiments, the freeze-drying as described in any one of the above successively includes the steps of pre-freezing, primary drying and secondary drying.

[0130] In some embodiments, the freeze-drying procedure is as follows: pre-freezing at a temperature of 5°C; pre-freezing at a temperature of -45°C; primary drying at a temperature of -20°C and a vacuum degree of 20 Pa; secondary drying at a temperature of 25°C and a vacuum degree of 1 Pa. In some embodiments, the freeze-drying procedure is as follows: pre-freezing at a temperature of 5°C for 10 min; pre-freezing at a temperature of -45°C for 50 min; primary drying at a temperature of -20°C and a vacuum degree of 20 Pa for 120 min; secondary drying at a temperature of 25°C and a vacuum degree of 1 Pa for 60 min.

[0131] In some embodiments, the lyophilized formulation is stable at 2 - 8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.

[0132] The present disclosure also provides a reconstituted form of the lyophilized formulation as described in any one of the above.

[0133] The present disclosure also provides a reconstituted solution containing an antibody - drug conjugate, wherein the reconstituted solution is obtained by reconstituting the lyophilized formulation as described in any one of the above.

[0134] In some embodiments, the reconstituted solution as described above comprises the following components:

[0135] (a) The anti - Claudin18.2 antibody - drug conjugate at a protein concentration of 10 mg / mL to 30 mg / mL, (b) Polysorbate at 0.1 mg / mL to 0.2 mg / mL, (c) Sugar at 40 mg / mL to 80 mg / mL, and (d) Histidine salt buffer at 10 mM to 30 mM; the pH of the reconstituted solution is about 5.0 - 5.5.

[0136] In some embodiments, the reconstituted solution as described above comprises the following components:

[0137] (a) The anti - Claudin18.2 antibody - drug conjugate at a protein concentration of 10 mg / mL to 30 mg / mL, (b) Polysorbate 80 at 0.1 mg / mL to 0.2 mg / mL, (c) Sucrose at 40 mg / mL to 80 mg / mL, and (d) Histidine salt buffer at 10 mM to 30 mM; the pH of the reconstituted solution is about 5.0 - 5.5.

[0138] In some embodiments, the reconstituted solution as described above comprises the following components:

[0139] (a) The anti - Claudin18.2 antibody - drug conjugate at a protein concentration of 20 mg / mL, (b) Polysorbate 80 at 0.2 mg / mL, (c) Sucrose at 40 mg / mL, and (d) 30 mM histidine - acetate buffer, and the pH of the reconstituted solution is 5.0 - 5.3.

[0140] The present disclosure also provides an article, which includes a container filled with the pharmaceutical composition as described in any one of the above, the lyophilized formulation as described in any one of the above, or the reconstituted solution as described in any one of the above.

[0141] The present disclosure also provides a method for treating a tumor or cancer, comprising administering to a subject an effective amount of the pharmaceutical composition as described in any one of the above, the lyophilized preparation as described in any one of the above, the reconstituted solution as described in any one of the above, or the article as described in any one of the above.

[0142] In some embodiments, the present disclosure also provides the use of the pharmaceutical composition as described in any one of the above, the lyophilized preparation as described in any one of the above, the reconstituted solution as described in any one of the above, or the article as described in any one of the above in the preparation of a medicament for treating a tumor or cancer.

[0143] In some embodiments, the present disclosure also provides the pharmaceutical composition as described in any one of the above, the lyophilized preparation as described in any one of the above, the reconstituted solution as described in any one of the above, or the article as described in any one of the above for use as a medicament.

[0144] In some embodiments, the tumor or cancer is preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krukenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis, and Merkel cell carcinoma.

[0145] In some embodiments, the lymphoma is selected from: Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma.

[0146] In some embodiments, the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer.

[0147] In some embodiments, the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia. Description of the Drawings

[0148] Figure 1 : FACS detection results of the binding of a humanized antibody to human Claudin18.2 at the cellular level.

[0149] Figure 2 : Endocytosis experiment of the humanized antibody in NUGC4 cells.

[0150] Figures 3A to 3C : Detection of the ADCC effect of the antibody in NUGC4 cells with different Claudin18.2 expression levels. Figure 3A is for the detection of the ADCC effect of the antibody in wild-type NUGC4 cells (low Claudin18.2 expression); Figure 3B is for the detection of the ADCC effect of the antibody in NUGC4 cells with medium Claudin18.2 expression; Figure 3C is for the detection of the ADCC effect of the antibody in NUGC4 cells with high Claudin18.2 expression.

[0151] Figure 4 : Antitumor experiment results of ADC-1 disclosed herein.

[0152] Figure 5 : Antitumor experiment results of ADC-2 disclosed herein. Detailed implementation manners

[0153] Terms

[0154] To make it easier to understand this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this disclosure pertains.

[0155] "Antibody-drug conjugate (ADC)" is a conjugate that links an antibody or an antibody fragment to a bioactive cytotoxin or a small molecule drug with cytotoxic activity through a stable chemical linker compound, taking full advantage of the specificity of the antibody for specifically binding to tumor cell-specific or highly expressed antigens and the high efficiency of the cytotoxin, and avoiding the toxic side effects on normal cells. Compared with traditional chemotherapy drugs in the past, antibody-drug conjugates can precisely bind to tumor cells and reduce the impact on normal cells.

[0156] "Buffer" refers to a buffer that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that control the pH within an appropriate range include acetate, succinate, gluconate, histidinate, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0157] "Histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include buffers such as histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, etc.; preferably histidine-acetate buffer. Histidine-acetate buffer is prepared from histidine and acetic acid, and histidine hydrochloride buffer is prepared from histidine and hydrochloric acid.

[0158] "Citrate buffer" is a buffer including citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, etc. The preferred citrate buffer is citric acid-sodium citrate.

[0159] "Succinate buffer" is a buffer including succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate salt, etc. The preferred succinate buffer is succinic acid-sodium succinate. Exemplarily, the said succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0160] "Phosphate buffer" is a buffer including phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, etc. The preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0161] "Acetate buffer" is a buffer including acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine acetate, acetic acid-potassium acetate, calcium acetate acetate, acetic acid-magnesium acetate, etc. The preferred acetate buffer is acetic acid-sodium acetate.

[0162] "Pharmaceutical composition" means a mixture containing one or more antibody-drug conjugates or their physiologically / pharmaceutically acceptable salts or prodrugs described herein and other chemical components, said other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to maintain the stability of the active ingredient, facilitate the administration to the organism, and facilitate the absorption of the active ingredient and thus exert its biological activity.

[0163] In this disclosure, "pharmaceutical composition" and "preparation" are not mutually exclusive.

[0164] In the solution form of the pharmaceutical composition described in this disclosure, unless otherwise specified, the solvent therein is water.

[0165] "Lyophilized preparation" means a preparation or pharmaceutical composition obtained after a vacuum freeze-drying step of a pharmaceutical composition or a liquid or solution preparation in liquid or solution form.

[0166] Although this disclosure provides content ranges or content values, those of ordinary skill in the art understand that the content ranges or content values cover the acceptable error ranges of the specific values measured.

[0167] The pharmaceutical compositions described in this disclosure can achieve a stable effect: a pharmaceutical composition in which the antibody-drug conjugate substantially retains its physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the predetermined shelf life of the pharmaceutical composition. Currently, there are various analytical techniques for measuring protein stability, which can measure the stability after storage at a selected temperature for a selected period of time.

[0168] A stable formulation is a formulation in which no significant changes are observed when stored at refrigerated temperatures (2 - 8 °C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Additionally, stable liquid formulations include those liquid formulations that exhibit desired characteristics after being stored for periods including 1 month, 3 months, 6 months at temperatures including 25 °C. Typical examples of stability: Measured by SEC-HPLC, generally no more than about 10%, preferably no more than about 5% of the antibody monomers aggregate or degrade. By visual analysis, the formulation is a pale yellow to nearly colorless clear liquid or colorless, or clear to slightly milky white. The concentration, pH, and osmolality of the formulation have a change of no more than ±10%. Generally, no more than about 10%, preferably no more than about 5% reduction is observed. Generally, no more than about 10%, preferably no more than about 5% aggregation is formed.

[0169] If, after visual inspection of color and / or clarity, or measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody-drug conjugate does not show a significant increase in aggregation, precipitation, and / or denaturation, then the antibody-drug conjugate "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0170] If the antibody-drug conjugate does not show significant chemical alterations, then the antibody "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be evaluated by detecting and quantifying the protein in its chemically altered forms. Degradation processes that often alter the protein chemical structure include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (evaluated by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement, etc.), and isomerization (evaluated by measuring the isoaspartate content, peptide mapping, etc.).

[0171] If the biological activity of the antibody-drug conjugate at a given time is within a predetermined range of the biological activity exhibited at the time of preparation of the pharmaceutical formulation, then the antibody-drug conjugate "retains its biological activity" in the pharmaceutical formulation.

[0172] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0173] The term "antibody" as used in this disclosure is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies or antigen-binding fragments thereof (also referred to as "antigen-binding portions"), provided that they exhibit the desired antigen-binding activity. A full-length antibody is an immunoglobulin (Ig) that comprises at least two heavy chains and two light chains that are inter-connected by disulfide bonds. The amino acid composition and arrangement of the constant regions of the immunoglobulin heavy chains are different, and thus their antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same class of Ig can be further divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are divided into κ chains or λ chains according to their constant regions. Each of the five classes of Ig can have κ chains or λ chains.

[0174] The sequences of approximately 110 amino acids near the N-terminus of the full-length antibody heavy and light chains vary greatly and are the variable regions (abbreviated as Fv regions); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant regions. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL). The heavy chain variable region and the light chain variable region include hypervariable regions (also known as complementarity-determining regions, abbreviated as CDR or HVR) and relatively conserved framework regions (also known as framework regions, abbreviated as FR). Each VL and VH consists of 3 CDRs and 4 FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0175] The "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region described in this disclosure refer to variants of the heavy chain constant region or the light chain constant region that are publicly available in the prior art and are derived from humans and do not change the structure and function of the antibody variable region. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants with site-directed modification and amino acid substitution of the heavy chain constant region. Specific substitutions such as the YTE mutation, L234A and / or L235A mutations, S228P mutation, 265A (such as D265A) and / or 297A (such as N297A), and / or mutations to obtain a knob-into-hole structure (such that the antibody heavy chain has a knob-Fc and hole-Fc combination), which have been shown to endow the antibody with new properties but do not change the function of the antibody variable region.

[0176] The term "antigen-binding fragment" or "functional fragment" or "antigen-binding portion" refers to one or more fragments of a full-length antibody that retain the ability to specifically bind an antigen. It has been shown that fragments of full-length antibodies can be used for the antigen-binding function of the antibody. Exemplary examples of binding fragments covered by the term "antigen-binding fragment" include: (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) F(ab') 2Fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region, (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VH and VL domains of a single arm of an antibody; (v) dsFv, a stable antigen-binding fragment formed by VH and VL via an interchain disulfide bond; (vi) diabodies, bispecific antibodies and multispecific antibodies comprising fragments such as scFv, dsFv, Fab, etc. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be joined by a recombinant method using an artificial peptide linker capable of forming them into a single protein chain, wherein VL and VH pair to form a monovalent molecule, called single-chain Fv (scFv) (see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0177] The term "amino acid difference" or "amino acid mutation" refers to an alteration or mutation in amino acids in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion or substitution of 1, 2, 3 or more amino acids based on the original protein or polypeptide.

[0178] The term "antibody framework" or "FR region" refers to a part of the variable domain VL or VH that serves as a scaffold for the antigen-binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

[0179] The terms "complementary determining region", "CDR", or "hypervariable region" refer to one of six hypervariable regions within the variable domain of an antibody that are primarily responsible for antigen binding. Typically, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3). The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (Lefranc M.P., Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003), etc. For example, for the classical format, following the Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following the Chothia rules, the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).In accordance with the IMGT rules, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align. In accordance with the AbM rules, the CDR amino acid numbering in VH is 26 - 32 (HCDR1), 50 - 58 (HCDR2), and 95 - 102 (HCDR3); and the amino acid residue numbering in VL is 24 - 34 (LCDR1), 50 - 56 (LCDR2), and 89 - 97 (LCDR3). The heavy chain variable region and the light chain variable region of the antibodies disclosed herein and their CDRs conform to the Kabat numbering rules.

[0180] "Amino acid sequence 'identity'" means, when amino acid sequences are aligned (introducing gaps if necessary to achieve the maximum percentage of sequence identity), the percentage of identical amino acid residues in the first sequence to those in the second sequence; where conservative substitutions are not considered part of sequence identity. To determine the percentage of amino acid sequence identity, the alignment can be achieved in a variety of ways within the skill in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0181] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified by conventional methods. For example, the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred prior art, mammalian expression systems result in glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing the antibody that binds to the antigen. The positive clones are expanded in serum-free medium in a bioreactor to produce the antibody. The culture broth secreting the antibody can be purified by conventional techniques. For example, purification can be performed using an A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble aggregates and multimers can also be removed by conventional methods, such as molecular sieving, ion exchange. The resulting product needs to be immediately frozen, such as at -70 °C, or lyophilized.

[0182] "Conservative modification" or "conservative substitution or replacement" refers to the substitution of an amino acid in a protein with another amino acid having similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.), such that the change can be made frequently without altering the biological activity of the protein. Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide basically does not change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., page 224, (4th edition)). Additionally, substitution of amino acids with similar structure or function is unlikely to disrupt the biological activity. Exemplary conservative substitutions are as follows:

[0183] Table a. Conservative substitutions of amino acids

[0184]

[0185]

[0186] The term "naked antibody" refers to an antibody that is not conjugated to a heterologous module (such as a cytotoxic module) or a radioactive label. In this disclosure, the content of the antibody-drug conjugate is measured by protein concentration, that is, by weight / volume of the protein (antibody part) in the conjugate.

[0187] The term "linker unit" or "linker" refers to a chemical structural fragment or bond that is connected to an antibody or its antigen-binding fragment at one end and to a drug at the other end, or can be connected to other linkers and then to the drug. Preferred embodiments of this disclosure are represented as L and L 1 to L 4 where L 1 end is connected to the antibody, and L 4 end is connected to the structural unit Y and then to a compound or toxin. Linkers, including extenders, spacers, and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker can be a "cleavable linker" that facilitates the release of the drug in cells. For example, acid-labile linkers (such as hydrazones), protease-sensitive (such as peptidase-sensitive) linkers, photo-labile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).

[0188] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched-chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof, etc. More preferably, it is a lower alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any available bonding point. The substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo group.

[0189] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein the alkyl group is as defined above.

[0190] The term "alkylene" refers to a saturated straight-chain or branched-chain aliphatic hydrocarbon group having two residues derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane. The alkylene is a straight-chain or branched-chain group containing 1 to 20 carbon atoms, preferably an alkylene containing 1 to 12 carbon atoms, and more preferably an alkylene containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 ))-), 1,2-ethylene (-CH 2 CH 2 ))-, 1,1-propylene (-CH(CH 2 CH 3 ))-), 1,2-propylene (-CH 2 CH(CH 3 ))-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -), and 1,5-butylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -), etc. The alkylene can be substituted or unsubstituted. When substituted, the substituent can be substituted at any available point of attachment, and the substituent is preferably independently optionally selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclic group, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo group.

[0191] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), where the definitions of alkyl or cycloalkyl are as described above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0192] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. The cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyls include spiro, fused, and bridged cycloalkyls.

[0193] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent that contains 3 to 20 ring atoms, one or more of which are heteroatoms selected from nitrogen, oxygen, or S(O) m (where m is an integer from 0 to 2), but does not include ring moieties of -O-O-, -O-S-, or -S-S-, and the remaining ring atoms are carbon. The heterocyclic group preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclics include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclics include spiro, fused, and bridged heterocyclics.

[0194] The term "spiroheterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which a single atom (called the spiro atom) is shared between monocyclic rings, one or more of which are heteroatoms selected from nitrogen, oxygen, or S(O) m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated π-electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members. Spiroheterocyclic groups are classified into monospiroheterocyclic groups, dispiroheterocyclic groups, or polyspiroheterocyclic groups according to the number of spiro atoms shared between rings, preferably monospiroheterocyclic groups and dispiroheterocyclic groups. More preferably, they are 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospiroheterocyclic groups. Non-limiting examples of spiroheterocyclic groups include:

[0195]

[0196] The term "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic system in which each ring shares a pair of adjacent atoms with other rings in the system. In a fused heterocyclic group, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π-electron system, and one or more ring atoms are selected from nitrogen, oxygen, or S(O) ma heteroatom (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. The fused heterocyclic group is preferably 6 to 14 membered, more preferably 7 to 10 membered. Depending on the number of constituent rings, the fused heterocyclic group can be classified as bicyclic, tricyclic, tetracyclic or polycyclic; preferably bicyclic or tricyclic; more preferably a 5-membered / 5-membered, or 5-membered / 6-membered bicyclic fused heterocyclic group. Non-limiting examples of the fused heterocyclic group include:

[0197]

[0198] The term "bridged heterocyclic group" refers to a polycyclic heterocyclic group having 5 to 14 members, wherein any two rings share two non-directly connected atoms. The bridged heterocyclic group may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system, and one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. The bridged heterocyclic group is preferably 6 to 14 membered, more preferably 7 to 10 membered. Depending on the number of constituent rings, the bridged heterocyclic group can be classified as bicyclic, tricyclic, tetracyclic or polycyclic; preferably bicyclic, tricyclic or tetracyclic; more preferably bicyclic or tricyclic. Non-limiting examples of the bridged heterocyclic group include:

[0199]

[0200] The heterocyclic group ring can be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is the heterocyclic group, and non-limiting examples thereof include:

[0201] etc.

[0202] The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo group.

[0203] The term "aryl" refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent carbon atom pairs) group having a conjugated π-electron system. The aryl is preferably 6 to 10 membered, such as phenyl and naphthyl, preferably phenyl. The aryl ring can be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is the aryl ring, and non-limiting examples thereof include:

[0204]

[0205] The aryl group can be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0206] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10 membered, more preferably 5 - or 6 - membered, such as furyl, thienyl, pyridyl, pyrrolyl, N - alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclic, or cycloalkyl ring, and the ring connected to the parent structure is the heteroaryl ring. Non - limiting examples include:

[0207]

[0208] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0209] The term "amino - protecting group" is a group that, when other parts of the molecule are reacting, protects the amino group with an easily removable group to keep the amino group unchanged. Non - limiting examples include 9 - fluorenylmethoxycarbonyl, tert - butoxycarbonyl, acetyl, benzyl, allyl, and p - methoxybenzyl, etc. These groups can be optionally substituted by 1 - 3 substituents selected from halogen, alkoxy, or nitro. The amino - protecting group is preferably 9 - fluorenylmethoxycarbonyl.

[0210] The term "cycloalkylalkyl" means that the hydrogen on the alkyl is substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, where the alkyl is as defined above and the cycloalkyl is as defined above.

[0211] The term "haloalkyl" means that the hydrogen on the alkyl is substituted by one or more halogen atoms, where the alkyl is as defined above.

[0212] The term "deuterated alkyl" means that the hydrogen on the alkyl is substituted by one or more deuterium atoms, where the alkyl is as defined above.

[0213] The term "hydroxy" refers to the - OH group.

[0214] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0215] The term "amino" refers to - NH2 。

[0216] The term "nitro" refers to -NO 2 。

[0217] "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and this description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1 - 3 variable regions of the antibody heavy chain" means that the variable regions of the antibody heavy chain of a specific sequence may, but need not, be present.

[0218] "Substituted" means that one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, are independently replaced by an appropriate number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) what substitutions are possible or impossible without undue effort. For example, an amino or hydroxyl group with a free hydrogen may be unstable when combined with a carbon atom having an unsaturated (such as olefinic) bond.

[0219] The term "drug - loading amount" refers to the average number of cytotoxic drugs loaded on each antibody or its antigen - binding fragment in an ADC molecule, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The range of drug - loading can be 0 - 12, preferably 1 - 10, more preferably 2 - 8, and most preferably 3.5 - 4.5 cytotoxic drugs (D) linked to each antibody or its antigen - binding fragment (Pc). In the embodiments disclosed herein, the drug - loading amount is represented as n, and exemplary n can be one or more calculated means among 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Conventional methods such as UV / visible light spectroscopy, mass spectrometry, ELISA assays, and HPLC can be used to characterize the average number of drugs in each ADC molecule after the conjugation reaction.

[0220] In one embodiment disclosed herein, the cytotoxic drug is conjugated to the N - terminal amino group, the ε - amino group of lysine residues, and / or the thiol group of an antibody or its antigen - binding fragment through a linker unit. Generally, the number of drug molecules that can be conjugated to the antibody in the conjugation reaction will be less than the theoretical maximum.

[0221] The drug - loading amount of the cytotoxic drug can be controlled by the following non - restrictive methods, including:

[0222] (1) Controlling the molar ratio of the coupling reagent and the monoclonal antibody,

[0223] (2) Controlling the reaction time and temperature,

[0224] (3) Selecting different reaction reagents.

[0225] The preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

[0226] The term "carrier" as used in the pharmaceutical compositions of the present disclosure refers to a system that can change the way a drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the target organ. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymeric surfactants that can serve as carriers can self-assemble due to their unique amphiphilic structure to form various forms of aggregates, and preferred examples include micelles, microemulsions, gels, liquid crystals, vesicles, etc. These aggregates have the ability to encapsulate drug molecules and at the same time have good permeability to membranes, and can serve as excellent drug carriers.

[0227] "Administer", "give", and "treat" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids mean the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administer", "give", and "treat" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes the contact of a reagent with the cells, as well as the contact of the reagent with a fluid, wherein the fluid contacts the cells. "Administer", "give", and "treat" also mean treatment of cells in vitro and ex vivo by a reagent, diagnostic, binding composition, or by another cell. "Treat" when applied to humans, veterinary medicine, or research subjects means therapeutic treatment, preventive or prophylactic measures, research, and diagnostic applications.

[0228] "Treat" means administering to a patient an internal or external therapeutic agent, such as a composition comprising any one of the binding compounds of the present disclosure, the patient having one or more disease symptoms, and the therapeutic agent being known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered to the treated patient or population in an amount effective to relieve one or more disease symptoms to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to relieve any specific disease symptom (also referred to as "therapeutically effective amount") can vary depending on various factors, such as the patient's disease state, age, and weight, as well as the ability of the drug to produce the desired effect in the patient. Whether a disease symptom has been alleviated can be evaluated by any clinical test method commonly used by a doctor or other professional healthcare person to evaluate the severity or progression of the symptom. Although the embodiments of the present disclosure (such as treatment methods or articles) may not be effective in relieving each target disease symptom, it should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test method known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0229] "Effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as, for example, the condition to be treated, the overall health of the patient, the method of administration, the route and dosage, and the severity of side effects. The effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0230] "Replacement" refers to the replacement of the solvent system of an antibody or ADC, for example, the replacement of a high-salt or hypertonic solvent system containing an antibody or ADC by a buffer system of a stable formulation through physical manipulation means, so that the antibody protein is present in the stable formulation. The so-called physical manipulation means include, but are not limited to, ultrafiltration, dialysis, or reconstitution after centrifugation.

[0231] Examples

[0232] The present disclosure is further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. For the experimental methods without specific conditions noted in the examples of the present disclosure, they are generally carried out under conventional conditions, such as referring to the "Antibody Technology Experimental Manual" and "Molecular Cloning Manual" published by Cold Spring Harbor Laboratory; or according to the conditions recommended by the raw material or commodity manufacturer. Reagents without specific sources noted are conventional reagents purchased from the market.

[0233] I. Antibody-drug conjugate

[0234] Preparation of anti-Claudin18.2 antibody

[0235] Example 1-1: Construction of a cell line highly expressing Claudin18.2

[0236] Using Lipofectamine 3000 transfection reagent, transfect the pCDH-hClaudin18.2 lentiviral expression vector plasmid and the pVSV-G or pCMV-dR8.91 lentiviral system packaging vector into the virus packaging cell 293T; collect the culture medium supernatant containing the virus, filter and perform ultra-high speed centrifugation; infect the human gastric signet ring cell carcinoma cell line NUGC4 with the concentrated virus, screen with puromycin for two to three weeks, and then perform FACS single cell sorting.

[0237] The expression level of Claudin18.2 is distinguished according to the tumor IHC score. Cells with a Claudin18.2 expression level equivalent to that of tumors with a tumor IHC score of 3 are high-expression cells, and cells with a Claudin18.2 expression level equivalent to that of tumors with a tumor IHC score of 2 are medium-expression cells.

[0238] According to the detection of Claudin18.2 expression on the surface of lentivirus-infected NUGC4 cells by FACS, NUGC4 / hClaudin18.2 monoclonal cell lines with high Claudin18.2 expression were selected. At the same time, the Claudin18.2 expression on the surface of wild-type NUGC4 cells was detected by FACS, and NUGC4 clone cell lines with medium Claudin18.2 expression were selected. Wild-type NUGC4 is a cell with low Claudin18.2 expression.

[0239] The selected monoclonal cell lines were expanded in culture and cryopreserved for subsequent experiments.

[0240] Claudin18.2 sequence Genbank: NP_001002026 (SEQ ID NO: 1): MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV.

[0241] Claudin18.2 DNA sequence (SEQ ID NO: 2):

[0242] AGAATTGCGC TGTCCACTTG TCGTGTGGCT CTGTGTCGAC ACTGTGCGCC ACCATGGCCG

[0243] TGACTGCCTG TCAGGGCTTG GGGTTCGTGG TTTCACTGAT TGGGATTGCG GGCATCATTG

[0244] CTGCCACCTG CATGGACCAG TGGAGCACCC AAGACTTGTA CAACAACCCC GTAACAGCTG

[0245] TTTTCAACTA CCAGGGGCTG TGGCGCTCCT GTGTCCGAGA GAGCTCTGGC TTCACCGAGT

[0246] GCCGGGGCTA CTTCACCCTG CTGGGGCTGC CAGCCATGCT GCAGGCAGTG CGAGCCCTGA

[0247] TGATCGTAGG CATCGTCCTG GGTGCCATTG GCCTCCTGGT ATCCATCTTT GCCCTGAAAT

[0248] GCATCCGCATTGGCAGCATG GAGGACTCTG CCAAAGCCAA CATGACACTG ACCTCCGGGA

[0249] TCATGTTCAT TGTCTCAGGT CTTTGTGCAA TTGCTGGAGT GTCTGTGTTT GCCAACATGC

[0250] TGGTGACTAA CTTCTGGATG TCCACAGCTA ACATGTACAC CGGCATGGGT GGGATGGTGC

[0251] AGACTGTTCA GACCAGGTAC ACATTTGGTG CGGCTCTGTT CGTGGGCTGG GTCGCTGGAG

[0252] GCCTCACACT AATTGGGGGT GTGATGATGT GCATCGCCTG CCGGGGCCTG GCACCAGAAG

[0253] AAACCAACTA CAAAGCCGTT TCTTATCATG CCTCAGGCCA CAGTGTTGCC TACAAGCCTG

[0254] GAGGCTTCAAGGCCAGCACT GGCTTTGGGT CCAACACCAA AAACAAGAAG ATATACGATG

[0255] GAGGTGCCCG CACAGAGGAC GAGGTACAAT CTTATCCTTC CAAGCACGAC TATGTGTAAT

[0256] GCTCTAAGACCTCTCAGCACGGGCGGAAGA AACTCCCGGA GAGCTCACCC AAAAAACAAG

[0257] GAGATCCCAT CTAGATTTCT TCTTGCTTTT GACTCACAGC TGGAAGTTAG AAAAGCCTCG

[0258] ATTTCATCTT TGGAGAGGCC AAATGGTCTT AGCCTCAGTC TCTGTCTCTA AATATTCCAC

[0259] CATAAAACAG CTGAGTTATT TATGAATTAG AGGCTATAGC TCACATTTTC AATCCTCTAT

[0260] TTCTTTTTTT AAATATAACT TTCTACTCTG ATGAGAGAAT GTGGTTTTAA TCTCTCTCTC

[0261] ACATTTTGAT GATTTAGACA GACTCCCCCT CTTCCTCCTA GTCAATAAAC CCATTGATGA

[0262] TCTATTTCCC AGCTTATCCC CAAGAAAACT TTTGAAAGGA AAGAGTAGAC CCAAAGATGT

[0263] TATTTTCTGC TGTTTGAATT TTGTCTCCCC ACCCCCAACT TGGCTAGTAA TAAACACTTA

[0264] CTGAAGAAGA AGCAATAAGA GAAAGATATT TGTAATCTCT CCAGCCCATG ATCTCGGTTT

[0265] TCTTACACTG TGATCTTAAA AGTTACCAAA CCAAAGTCAT TTTCAGTTTG AGGCAACCAA

[0266] ACCTTTCTAC TGCTGTTGAC ATCTTCTTAT TACAGCAACA CCATTCTAGG AGTTTCCTGA

[0267] GCTCTCCACT GGAGTCCTCT TTCTGTCGCG GGTCAGAAAT TGTCCCTAGA TGAATGAGAA

[0268] AATTATTTTT TTTAATTTAA GTCCTAAATA TAGTTAAAAT AAATAATGTT TTAGTAAAAT

[0269] GATACACTAT CTCTGTGAAA TAGCCTCACC CCTACATGTG GATAGAAGGA AATGAAAAAA

[0270] TAATTGCTTT GACATTGTCT ATATGGTACT TTGTAAAGTC ATGCTTAAGT ACAAATTCCA

[0271] TGAAAAGCTC ACTGATCCTA ATTCTTTCCC TTTGAGGTCT CTATGGCTCT GATTGTACAT

[0272] GATAGTAAGT GTAAGCCATG TAAAAAGTAA ATAATGTCTG GGCACAGTGG CTCACGCCTG

[0273] TAATCCTAGCACTTTGGGAG GCTGAGGAGG AAGGATCACT TGAGCCCAGA AGTTCGAGAC

[0274] TAGCCTGGGCAACATGGAGAAGCCCTGTCT CTACAAAATA CAGAGAGAAA AAATCAGCCA

[0275] GTCATGGTGG CCTACACCTG TAGTCCCAGC ATTCCGGGAG GCTGAGGTGG GAGGATCACT

[0276] TGAGCCCAGGGAGGTTGGGG CTGCAGTGAG CCATGATCAC ACCACTGCAC TCCAGCCAGG

[0277] TGACATAGCGAGATCCTGTC TAAAAAAATA AAAAATAAAT AATGGAACAC AGCAAGTCCT

[0278] AGGAAGTAGGTTAAAACTAA TTCTTTAAAA AAAAAAAAAA GTTGAGCCTG AATTAAATGT

[0279] AATGTTTCCA AGTGACAGGT ATCCACATTT GCATGGTTAC AAGCCACTGC CAGTTAGCAG

[0280] TAGCACTTTC CTGGCACTGT GGTCGGTTTT GTTTTGTTTT GCTTTGTTTA GAGACGGGGT

[0281] CTCACTTTCC AGGCTGGCCT CAAACTCCTG CACTCAAGCA ATTCTTCTAC CCTGGCCTCC

[0282] CAAGTAGCTG GAATTACAGG TGTGCGCCAT CACAACTAGC TGGTGGTCAG TTTTGTTACT

[0283] CTGAGAGCTG TTCACTTCTC TGAATTCACC TAGAGTGGTT GGACCATCAG ATGTTTGGGC

[0284] AAAACTGAAA GCTCTTTGCA ACCACACACC TTCCCTGAGC TTACATCACT GCCCTTTTGA

[0285] GCAGAAAGTC TAAATTCCTT CCAAGACAGT AGAATTCCAT CCCAGTACCA AAGCCAGATA

[0286] GGCCCCCTAGGAAACTGAGG TAAGAGCAGT CTCTAAAAAC TACCCACAGC AGCATTGGTG

[0287] CAGGGGAACT TGGCCATTAG GTTATTATTT GAGAGGAAAG TCCTCACATC AATAGTACAT

[0288] ATGAAAGTGACCTCCAAGGG GATTGGTGAA TACTCATAAG GATCTTCAGG CTGAACAGAC

[0289] TATGTCTGGG GAAAGAACGG ATTATGCCCC ATTAAATAAC AAGTTGTGTT CAAGAGTCAG

[0290] AGCAGTGAGCTCAGAGGCCC TTCTCACTGA GACAGCAACA TTTAAACCAA ACCAGAGGAA

[0291] GTATTTGTGG AACTCACTGC CTCAGTTTGG GTAAAGGATG AGCAGACAAG TCAACTAAAG

[0292] AAAAAAGAAAAGCAAGGAGGAGGGTTGAGC AATCTAGAGC ATGGAGTTTG TTAAGTGCTC

[0293] TCTGGATTTG AGTTGAAGAG CATCCATTTG AGTTGAAGGC CACAGGGCAC AATGAGCTCT

[0294] CCCTTCTACC ACCAGAAAGT CCCTGGTCAG GTCTCAGGTA GTGCGGTGTG GCTCAGCTGG

[0295] GTTTTTAATT AGCGCATTCT CTATCCAACA TTTAATTGTT TGAAAGCCTC CATATAGTTA

[0296] GATTGTGCTT TGTAATTTTG TTGTTGTTGC TCTATCTTAT TGTATATGCA TTGAGTATTAACCTGAATGT TTTGTTACTT AAATATTAAA AACACTGTTA TCCTACAGTT。

[0297] Example 1-2: Generation of anti-human claudin18.2 monoclonal antibody

[0298] 1 Immunization

[0299] Generate anti-human Claudin18.2 monoclonal antibody by immunizing mice.

[0300] SJL white mice were used in the experiment. They were female, 6 - 8 weeks old (Charles River Laboratory Animal Technology Co., Ltd., Beijing, license number for animal production: SCXK(Beijing)2012 - 0001). Breeding environment: SPF level. After the mice were purchased, they were raised in the laboratory environment for 1 week, with a 12 / 12 - hour light / dark cycle adjustment, temperature at 20 - 25 °C, and humidity at 40 - 60%. The mice that had adapted to the environment were immunized according to the following protocol. The immunization antigen was huClaudin18.2 - HEK293 cells (HEK - 293 stable transfected cell line transfected with human Claudin18.2 plasmid).

[0301] Immunization protocol: Before the first immunization with cells, Gold Adjuvant (Sigma Cat No.T2684) at 0.1 ml / mouse was injected intraperitoneally (IP); half an hour later, 0.1 ml of cell suspension diluted to a concentration of 1×10 8 / ml with normal saline was injected intraperitoneally (IP) into each mouse. After the cells were evenly resuspended, inoculation was carried out at days 0, 14, 28, 42, and 56. Blood was taken at days 21, 35, 49, and 63, and the antibody titer in the mouse serum was determined by ELISA method. After the 4th - 5th immunization, mice with high antibody titers in the serum and the titers tending to reach a plateau were selected for spleen cell fusion. Three days before spleen cell fusion, booster immunization was carried out by injecting 1×10 7 cells intraperitoneally (IP).

[0302] 2 Spleen cell fusion

[0303] Spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells ( CRL - 8287 TM ) using PEG - mediated fusion steps to obtain hybridoma cells. The hybridoma cells were resuspended at a density of 0.5 - 1×10 6 / ml with complete medium (IMDM medium containing 20% FBS, 1×HAT, and 1×OPI), and 100 μl / well was seeded into 96 - well plates. Incubated at 37 °C, 5% CO 2 for 3 - 4 days, then 100 μl / well of HAT complete medium was added, and continued to culture for 3 - 4 days until clones formed. The supernatant was removed, and 200 μl / well of HT complete medium (IMDM medium containing 20% FBS, 1×HT, and 1×OPI) was added, and cultured at 37 °C, 5% CO 2 for 3 days, followed by ELISA detection.

[0304] 3 Hybridoma cell screening

[0305] According to the growth density of hybridoma cells, the culture supernatant was detected by the combined ELISA method. Cells with strong binding ability to huClaudin18.2-HEK293 cells and no binding to HEK293 cells were selected and amplified and cryopreserved in a timely manner; after two to three subclonings until single cell clones were obtained.

[0306] Cell binding experiments were required for each subcloned cell. Hybridoma clones were obtained through the above experiments, and antibodies were further prepared by serum-free cell culture method. The antibodies were purified according to the purification examples and used in the detection examples.

[0307] Examples 1-3: Humanization of murine antibodies

[0308] Monoclonal hybridoma cell lines mAb1901 and mAb1902 with high in vitro activity were selected; the monoclonal antibody sequences were cloned, and then humanized, recombinantly expressed and evaluated for activity.

[0309] The process of cloning the sequence from the hybridoma was as follows. Hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) (according to the steps of the kit instructions) and reverse transcribed (PrimeScript TM Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and sent to a sequencing company for sequencing. The amino acid sequences corresponding to the obtained DNA sequences are shown in SEQ ID NO: 3-6:

[0310] Murine heavy chain variable region of mAb1901 (SEQ ID NO: 3) EVQLMESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEMGLEWIAYISR

[0311] GSSTIYYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARGGYDTRNAMDYWGQGTSVTVSS;

[0312] Murine light chain variable region of mAb1901 (SEQ ID NO: 4)

[0313] DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLAIYHCQNDLYYPLTFGAGTKLELK;

[0314] mAb1902 murine heavy chain variable region (SEQ ID NO: 5)

[0315] EVQLQESGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSSSTAYMQLSSLPSEDSAVYYCARLKTGNSFDYWGQGTTLTVSS;

[0316] mAb1902 murine light chain variable region (SEQ ID NO: 6)

[0317] DIVLTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNAYTYPFTFGSGTKLEIK;

[0318] The above murine heavy chain variable region and light chain variable region are respectively linked to the heavy chain constant region of the following human IgG1 antibody and the human κ light chain constant region to form chimeric antibodies ch1901 and ch1902.

[0319] The constant region of each antibody is selected from the following sequences:

[0320] Heavy chain constant region of human IgG1 antibody: (SEQ ID NO: 7)

[0321] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0322] Human κ light chain constant region: (SEQ ID NO: 8)

[0323] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0324] As described in many documents in the art, the murine monoclonal antibody was humanized. Briefly, the murine constant domain was replaced with a human constant domain, and the human germline antibody sequence was selected based on the homology between the murine antibody and the human antibody for CDR grafting. In the present invention, candidate molecules with good activity were selected for humanization, and the results are as follows.

[0325] 1. CDR regions of murine antibody

[0326] The amino acid residues of VH / VL CDR in Table 1 were determined and annotated by the Kabat numbering system.

[0327] The CDR sequences of the murine antibody are as described in Table 1:

[0328] Table 1. CDR sequences of murine antibody

[0329] antibody mAb1901 HCDR1 DYGIH (SEQ ID NO: 9) HCDR2 YISRGSSTIYYADTVKG (SEQ ID NO: 10) HCDR3 GGYDTRNAMDY (SEQ ID NO: 11) LCDR1 KSSQSLLNSGNQKNYLA (SEQ ID NO: 12) LCDR2 GASTRAS (SEQ ID NO: 13) LCDR3 QNDLYYPLT (SEQ ID NO: 14) antibody mAb1902 HCDR1 SYWMH (SEQ ID NO: 15) HCDR2 MIHPNSGSTNYNEKFKG (SEQ ID NO: 16) HCDR3 LKTGNSFDY (SEQ ID NO: 17) LCDR1 KSSQSLLNSGNQKNYLT (SEQ ID NO: 18) LCDR2 WASTRES (SEQ ID NO: 19) LCDR3 QNAYTYPFT (SEQ ID NO: 20)

[0330] 2. Selection of human germline FR region sequences

[0331] Based on the obtained typical structure of the murine VH / VL CDR of the antibody, the heavy and light chain variable region sequences were compared with the antibody Germline database to obtain a highly homologous human germline template. Among them, the human germline light chain framework region is from the human κ light chain gene.

[0332] 2.1 Humanization and back mutation design of mAb1901

[0333] An appropriate human antibody germline was selected to humanize the murine antibody mAb1901. The CDR regions of the murine antibody mAb1901 were transplanted onto the selected humanized template to replace the humanized variable regions, and then recombined with the IgG constant region to form a complete antibody. At the same time, back mutations were performed on the FR regions in the V region of the humanized antibody. Exemplary back mutation methods and combinations are as follows:

[0334] Table 2. Humanized antibody of mAb1901 and its back mutations *

[0335]

[0336] *All amino acid position numbers in the table are numbered according to the Kabat numbering rule. In N82T of the heavy chain variable region, 82 is the 82A position according to the Kabat rule.

[0337] Table 3. Sequences of the light chain variable region and heavy chain variable region of the humanized antibody of mAb1901

[0338]

[0339] In the above table, the corresponding heavy chain variable region is linked to the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of the full-length antibody, and the light chain variable region is linked to the human κ light chain constant region shown in SEQ ID NO: 8 to form the light chain of the full-length antibody. In other embodiments, the heavy chain variable region and the light chain variable region can also be linked to other heavy chain constant regions and light chain constant regions respectively to form the full-length antibody.

[0340] 2.2 Humanization and back mutation design of mAb1902

[0341] An appropriate human antibody germline was selected to humanize the murine antibody mAb1902. The CDR regions of the murine antibody mAb1902 were transplanted onto the selected humanized template to replace the humanized variable regions, and then recombined with the IgG constant region to form a complete antibody. At the same time, back mutations were performed on the FR regions in the V region of the humanized antibody. Exemplary back mutation methods and combinations are as follows:

[0342] Table 4. Humanized antibody of mAb1902 and its back mutation design *

[0343]

[0344] *All amino acid position numbers in the table are numbered according to the Kabat numbering rule.

[0345] Table 5. Sequences of the light chain variable region and heavy chain variable region of the humanized antibody mAb1902

[0346]

[0347] In the above table, the corresponding heavy chain variable region is linked to the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of the full-length antibody, and the light chain variable region is linked to the human κ light chain constant region shown in SEQ ID NO: 8 to form the light chain of the full-length antibody.

[0348] Chimeric antibody ch1901

[0349] ch1901 heavy chain: (SEQ ID NO: 35)

[0350] EVQLMESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEMGLEWIAYISR

[0351] GSSTIYYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARGGYDTRN

[0352] AMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

[0353] SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

[0354] DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0355] SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

[0356] YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

[0357] PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0358] ch1901 light chain: (SEQ ID NO: 36)

[0359] DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLI

[0360] YGASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLAIYHCQNDLYYPLTFGAGTK

[0361] LELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0362] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0363] Chimeric antibody ch1902

[0364] ch1902 heavy chain: (SEQ ID NO: 37)

[0365] EVQLQESGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGMIHP

[0366] NSGSTNYNEKFKGKATLTLDKSSSTAYMQLSSLPSEDSAVYYCARLKTGNSFDY

[0367] WGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS

[0368] GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV

[0369] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED

[0370] PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC

[0371] KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI

[0372] AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0373] Light chain of ch1902: (SEQ ID NO: 38)

[0374] DIVLTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLI

[0375] YWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNAYTYPFTFGSGTK

[0376] LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0377] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0378] Table 6. Humanized antibody of mAb1901

[0379] heavy and light chains H1 H2 H3 H4 L1 h1901-1 h1901-2 h1901-3 h1901-4 L2 h1901-5 h1901-6 h1901-7 h1901-8 L3 h1901-9 h1901-10 h1901-11 h1901-12

[0380] The full-length antibody light and heavy chain sequences are shown below:

[0381] Table 7. Light and heavy chain sequences of humanized antibody of mAb1901

[0382]

[0383]

[0384]

[0385] Table 8. Humanized antibody of mAb1902

[0386] heavy and light chains H11 H12 H13 H14 L11 h1902-1 h1902-2 h1902-3 h1902-4 L12 h1902-5 h1902-6 h1902-7 h1902-8 L13 h1902-9 h1902-10 h1902-11 h1902-12

[0387] The full-length antibody light and heavy chain sequences are shown below:

[0388] Table 9. mAb1901 humanized antibody light and heavy chain sequences

[0389]

[0390]

[0391] The positive control antibody for this disclosure is IMAB-362 (from WO2016166122).

[0392] Heavy chain (SEQ ID NO: 53)

[0393] QVQLQQPGAE LVRPGASVKL SCKASGYTFT SYWINWVKQR PGQGLEWIGN

[0394] IYPSDSYTNY NQKFKDKATL TVDKSSSTAY MQLSSPTSED SAVYYCTRSW

[0395] RGNSFDYWGQ GTTLTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY

[0396] FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI

[0397] CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD

[0398] TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST

[0399] YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY

[0400] TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSKLTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK;

[0401] Light chain (SEQ ID NO: 54)

[0402] DIVMTQSPSS LTVTAGEKVT MSCKSSQSLL NSGNQKNYLT WYQQKPGQPP

[0403] KLLIYWASTR ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCQNDYSY

[0404] PFTFGSGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA

[0405] KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSPVTKSFNRGEC。

[0406] Clone, express, and purify the above antibodies using conventional gene cloning and recombinant expression methods, respectively.

[0407] Preparation of Anti-Claudin18.2 ADC Conjugates

[0408] drug

[0409] The drug moiety of the anti-Claudin18.2 ADC conjugates in this disclosure can be any suitable drug. Particularly suitable drugs are described, for example, in PCT Publication No. WO2020063676A1 (incorporated herein by reference in its entirety). Compound 9-A of this disclosure is N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide, which has the following structure:

[0410]

[0411] Analysis of drug loading in ADC bulk drug

[0412] 1. UV-HPLC Method

[0413] After placing the cuvette filled with sodium succinate buffer in the reference absorption cell and the sample measurement absorption cell respectively, after deducting the solvent blank, then place the cuvette filled with the sample solution to be measured in the sample measurement absorption cell, and measure the absorbance at 280 nm and 370 nm.

[0414] Result calculation: The loading amount of the ADC stock solution was determined by ultraviolet spectrophotometry (instrument used: Thermo nanodrop2000 ultraviolet spectrophotometer). The principle is that the total absorbance value of the ADC stock solution at a certain wavelength is equal to the sum of the absorbance values of the drug and the monoclonal antibody at that wavelength, that is:

[0415] (1) A 280nm =ε mab-280 bC mab +ε Drug-280 bC Drug

[0416] ε Drug-280 : The average molar extinction coefficient of the drug at 280 nm is 5100;

[0417] C Drug : The concentration of the drug;

[0418] ε mab-280 : The average molar extinction coefficient of the single antigen stock solution at 280 nm is 214600;

[0419] C mab : The concentration of the single antigen stock solution;

[0420] b: The optical path length is 1 cm.

[0421] Similarly, the total absorbance value equation of the sample at 370 nm can be obtained:

[0422] (2) A 370nm =ε mab-370 bC mab +ε Drug-370 bC Drug

[0423] ε Drug-370 : The average molar extinction coefficient of the drug at 370 nm is 19000;

[0424] C Drug : The concentration of the drug;

[0425] ε mab-370 : The extinction coefficient of the single antigen stock solution at 370 nm is 0;

[0426] C mab : The concentration of the single antigen stock solution;

[0427] b: The optical path length is 1 cm.

[0428] The drug loading can be calculated by combining the extinction coefficient and concentration data of the monoclonal antibody and the drug at two detection wavelengths using equations (1) and (2).

[0429] Drug loading = C Drug / C mab .

[0430] 2. RP-HPLC method

[0431] For the naked antibody and the ADC sample to be tested (concentration 1 mg / ml), add 4 μl of DDT (sigma) for reduction, incubate in a water bath at 37 °C for 1 hour, and then take it out and transfer it to an inner cannula. Use an Agilent 1200 high-performance liquid chromatograph for detection. The chromatographic column is an Agilent PLRP-S 1000A 8 μm 4.6 * 250 mm, column temperature: 80 °C; DAD detector wavelength 280 nm; flow rate: 1 mL / min; injection volume: 40 μL; then, by comparing the chromatograms of the sample and the naked antibody, distinguish the positions of the light and heavy chains, and then integrate the chromatogram of the test sample to calculate the DAR value.

[0432] Solution preparation:

[0433] 1) 0.25 M DTT solution:

[0434] Preparation example: Take 5.78 mg of DTT, add 150 μl of purified water and dissolve it fully to obtain 0.25 M DTT solution, and store it at -20 °C.

[0435] 2) Mobile phase A (0.1% aqueous TFA solution):

[0436] Preparation example: Measure 1000 ml of purified water with a measuring cylinder, add 1 mL of TFA (sigma), mix well and use, and store it at 2 - 8 °C for 14 days.

[0437] 3) Mobile phase B (0.1% acetonitrile solution of TFA):

[0438] Preparation example: Measure 1000 ml of acetonitrile with a measuring cylinder, add 1 mL of TFA, mix well and use, and store it at 2 - 8 °C for 14 days.

[0439] Data analysis:

[0440] By comparing the chromatograms of the sample and the naked antibody, distinguish the positions of the light and heavy chains, and then integrate the chromatogram of the test sample to calculate the DAR value.

[0441] The calculation formula is as follows:

[0442] Table 10. Drug-loading labeling table for ADC light and heavy chains

[0443] Name Number of conjugated drugs LC 0 LC + 1 2 HC 0 HC + 1 2 HC + 2 4 HC + 3 6

[0444] Total LC peak area = LC peak area + LC+1 peak area;

[0445] Total HC peak area = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area;

[0446] LC DAR = Σ(number of conjugated drugs * percentage of peak area) / Total LC peak area;

[0447] HC DAR = Σ(number of conjugated drugs * percentage of peak area) / Total HC peak area;

[0448] DAR = LC DAR + HC DAR.

[0449] Examples 1 - 4: ADC-1 / ADC-2

[0450] Under the condition of 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.03 mL, 110.3 μmol) was added to a PBS buffer solution containing antibody h1902-5 (PBS buffer solution with pH = 6.5, 0.05 M; 10.0 mg / mL, 320.0 mL, 21.62 μmol). The mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours, then the reaction was stopped.

[0451] The reaction solution was cooled to 25°C in a water bath. Then, compound 9-A (350 mg, 303 μmol) was dissolved in 13.2 ml of acetonitrile and 6.6 ml of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and reacted with shaking at 25°C for 3 hours, then the reaction was stopped.

[0452] The reaction solution was purified through an ultrafiltration membrane to remove small molecules. The purification was successively carried out using 5 L of 50 mM PBS buffer solution with pH = 6.5 (4% acetonitrile, 2% DMSO) and 5 L of 10 mM succinic acid buffer solution with pH = 5.3. Subsequently, sucrose was added to the purified solution to a concentration of 60 mg / mL and Tween-20 to a concentration of 0.2 mg / mL to prepare ADC-1 (succinic acid buffer solution with 10 mM, pH = 5.3; 10 mg / mL, 2.626 g), and the yield was 81.81%. Then, it was made into freeze-dried powder at 20 mg per vial.

[0453] The average value was calculated by UV-HPLC: n = 6.8.

[0454] Using the above method, ADC-2 can be prepared by using antibody h1901-11 instead of h1902-5 and compound 9-A, and n = 7.1.

[0455] Examples 1-5: ADC-3

[0456] At 37 °C, add the prepared aqueous TCEP solution (10 mM, 10.1 μL, 101 nmol) to the PBS buffer aqueous solution containing antibody h1901-11 (0.05 M PBS buffer aqueous solution with pH = 6.5; 10.0 mg / mL, 1 mL, 67.5 nmol), place it in a water bath oscillator, and react with shaking at 37 °C for 3 hours to stop the reaction. Cool the reaction solution to 25 °C using a water bath.

[0457] Dissolve compound 9-A (0.58 mg, 540 nmol) in 34 μl of DMSO, add it to the above reaction solution, place it in a water bath oscillator, and react with shaking at 25 °C for 3 hours to stop the reaction. Desalt and purify the reaction solution using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH = 6.5, containing 0.001 M EDTA) to obtain the PBS buffer solution of ADC-3 (0.72 mg / mL, 11.2 mL), and store it at 4 °C. Calculate the average value by RP-HPLC: n = 2.51.

[0458] Examples 1-6: ADC-4

[0459] At 37 °C, add the prepared aqueous TCEP solution (10 mM, 16.9 μL, 169 nmol) to the PBS buffer aqueous solution containing antibody h1901-11 (0.05 M PBS buffer aqueous solution with pH = 6.5; 10.0 mg / mL, 1 mL, 67.5 nmol), place it in a water bath oscillator, and react with shaking at 37 °C for 3 hours to stop the reaction. Cool the reaction solution to 25 °C using a water bath.

[0460] Dissolve compound 9-A (0.73 mg, 680 nmol) in 43 μl of DMSO, add it to the above reaction solution, place it in a water bath oscillator, and react with shaking at 25 °C for 3 hours to stop the reaction. Desalt and purify the reaction solution using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH = 6.5, containing 0.001 M EDTA) to obtain the PBS buffer solution of ADC-4 (0.62 mg / mL, 12.5 mL), and store it at 4 °C. Calculate the average value by RP-HPLC: n = 4.06.

[0461] Examples 1-7: ADC-5

[0462] At 37 °C, add the prepared aqueous TCEP solution (10 mM, 35.8 μL, 358 nmol) to the PBS buffer aqueous solution of antibody h1901-11 (0.05 M PBS buffer aqueous solution with pH = 6.5; 10.0 mg / mL, 1 mL, 67.5 nmol). Place it in a water bath oscillator and react with shaking at 37 °C for 3 hours to stop the reaction. Cool the reaction solution to 25 °C using a water bath.

[0463] Dissolve compound 9-A (1.09 mg, 1015 nmol) in 64 μl DMSO, add it to the above reaction solution, place it in a water bath oscillator, and react with shaking at 25 °C for 3 hours to stop the reaction. Desalt and purify the reaction solution using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH = 6.5, containing 0.001 M EDTA) to obtain the PBS buffer solution of ADC-5 (0.54 mg / mL, 12.5 mL), and store it at 4 °C. Calculate the average value by RP-HPLC: n = 6.8.

[0464] Example 1-8: ADC-6

[0465] At 37 °C, add the prepared aqueous TCEP solution (10 mM, 10.9 μL, 109 nmol) to the PBS buffer aqueous solution of antibody h1902-5 (0.05 M PBS buffer aqueous solution with pH = 6.5; 10.0 mg / mL, 1.08 mL, 72.9 nmol). Place it in a water bath oscillator and react with shaking at 37 °C for 3 hours to stop the reaction. Cool the reaction solution to 25 °C using a water bath.

[0466] Dissolve compound 9-A (0.63 mg, 587 nmol) in 40 μl DMSO, add it to the above reaction solution, place it in a water bath oscillator, and react with shaking at 25 °C for 3 hours to stop the reaction. Desalt and purify the reaction solution using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH = 6.5, containing 0.001 M EDTA) to obtain the PBS buffer solution of ADC-6 (0.7 mg / mL, 13.0 mL), and store it at 4 °C. Calculate the average value by RP-HPLC: n = 2.69.

[0467] Example 1-9: ADC-7

[0468] At 37 °C, to the PBS buffer aqueous solution of antibody h1902-5 (0.05 M PBS buffer aqueous solution with pH = 6.5; 10.0 mg / mL, 1.08 mL, 72.9 nmol) was added the prepared TCEP aqueous solution (10 mM, 18.3 μL, 183 nmol). It was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0469] Compound 9-A (0.79 mg, 736 nmol) was dissolved in 50 μl DMSO and added to the above reaction solution. It was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH = 6.5, containing 0.001 M EDTA) to obtain the PBS buffer solution of ADC-7 (0.6 mg / mL, 14.0 mL), which was stored at 4 °C. The average value was calculated by RP-HPLC: n = 4.25.

[0470] Example 1-10: ADC-8

[0471] At 37 °C, to the PBS buffer aqueous solution of antibody h1902-5 (0.05 M PBS buffer aqueous solution with pH = 6.5; 10.0 mg / mL, 1.08 mL, 72.9 nmol) was added the prepared TCEP aqueous solution (10 mM, 38.7 μL, 387 nmol). It was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath.

[0472] Compound 9-A (1.18 mg, 1099 nmol) was dissolved in 70 μl DMSO and added to the above reaction solution. It was placed in a water bath shaker and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH = 6.5, containing 0.001 M EDTA) to obtain the PBS buffer solution of ADC-8 (0.56 mg / mL, 14.2 mL), which was stored at 4 °C. The average value was calculated by RP-HPLC: n = 7.01.

[0473] Example 1-11: ADC-9

[0474] At 12 °C, the prepared TCEP histidine buffer (10 mM histidine buffer; 1.717 mM, 1.16 L, 1.99 mmol) was added to the histidine-acetate-Tris / EDTA buffer (buffer of 10 mM histidine-acetate-Tris and 2.5 mM EDTA at pH 7.2; 20.6 g / L, 6.49 L, 0.91 mmol) containing antibody h1902-5. It was placed in a constant temperature water bath and stirred at 12 °C for 2 hours. The reaction was stopped to obtain the intermediate I solution.

[0475] Compound 9-A (4.72 g, 4.39 mmol) was dissolved in 0.38 L of DMSO to form a DMSO solution of compound 9-A. 0.38 L of DMSO was pre-added to the above intermediate I solution, and then the DMSO solution of the above compound 9-A was added. It was placed in a constant temperature water bath and stirred at 12 °C for 1 hour. The reaction was stopped.

[0476] The above reaction solution was purified by a Capto S Impact cation exchange chromatography column, washed with 9 column volumes of 0.05 M acetate buffer (pH = 5.0) containing 10% (v / v) DMSO and 6 column volumes of 0.05 M acetate buffer (pH = 5.0), and then eluted with 0.05 M acetate, 0.30 M sodium chloride buffer (pH = 5.5) to remove free toxins and residual solvents in the reaction solution. At 22 °C, the cation eluate was ultrafiltered with 7 times the volume (the ultrafiltration membrane package used a 30 KD polycellulose membrane package) to obtain the product ADC-9. RP-HPLC calculation average value: n = 4.1.

[0477] Biological evaluation

[0478] Test example 1: Cell-level ELISA binding experiment

[0479] Cell-based ELISA experiments were used to detect the binding characteristics of Claudin18.2 antibodies. NUGC4 cells stably expressing Claudin18.2 were cultured in 96-well cell plates (Corning, 3599). When the cells grew to 90% confluence, 4% paraformaldehyde was added to fix the cells for 1 hour. After washing the plates 3 times with PBST buffer (PBS with 0.05% Tween-20, pH 7.4), 200 μl / well of 5% skim milk (Bright skim milk powder) blocking solution diluted with PBS was added, and the plates were incubated in a 37°C incubator for 2.5 hours or left overnight (16 - 18 hours) at 4°C for blocking. After the blocking was completed, the blocking solution was discarded, and the plates were washed 3 times with PBST buffer. Then, 50 μl / well of different concentrations of the antibody to be tested diluted with sample dilution buffer (PBS with 1% skim milk, pH 7.4) was added, and the plates were incubated in a 37°C incubator for 2 hours. After the incubation, the plates were washed 5 times with PBST, and 100 μl / well of HRP-labeled goat anti-human secondary antibody (Jackson Immuno Research, 109 - 035 - 003) diluted with sample dilution buffer was added, and the plates were incubated at 37°C for 1 hour. After washing the plates 6 times with PBST, 50 μl / well of TMB chromogenic substrate (KPL, 52 - 00 - 03) was added, and the plates were incubated at room temperature for 10 - 15 min. Then, 50 μl / well of 1M H 2 SO 4 The reaction was terminated, and the absorbance was read at 450 nm using an MD Versa Max TM microplate reader to calculate the binding EC50 value of the Claudin18.2 antibody to Claudin18.2.

[0480] Table 11. Binding activity of antibodies

[0481] antibody IMAB362 ch1901 ch1902 Emax 1.175 1.399 1.272 EC50 (nM) 0.108 0.098 0.074

[0482] Table 12. Binding activity of mAb1901 humanized antibody

[0483] antibody Emax EC50 (nM) IMAB362 1.115 0.086 h1901-2 1.039 0.076 h1901-3 1.1055 0.22 h1901-4 0.986 0.201 h1901-6 0.937 0.091 h1901-7 0.921 0.166 h1901-8 1.047 0.091 h1901-11 1.44 0.076 h1901-12 1.22 0.116

[0484] Table 13. Binding activity of mAb1902 humanized antibody

[0485] antibody Emax EC50 (nM) IMAB362 0.88 0.187 h1902-1 0.87 0.113 h1902-2 0.88 0.107 h1902-3 0.84 0.175 h1902-4 0.82 0.087 h1902-5 0.9 0.098 h1902-6 0.78 0.141 h1902-7 0.75 0.121 h1902-8 0.89 0.132 h1902-9 0.75 0.137 h1902-10 0.89 0.133

[0486] Test Example 2: Antibody binding experiment at the cellular level

[0487] NUGC4 cells stably expressing Claudin18.2 were prepared into 1×10 6Cell suspension at / ml, 100 μl per well was added into a 96-well round bottom plate (Corning, 3795). After centrifugation to remove the supernatant, different concentrations of the test Claudin18.2 antibody diluted with FACS buffer at 50 μl per well were added, and it was incubated in the dark at 4°C for 1 hour. After centrifugal washing 3 times with FACS buffer at 300 g, Alexa Fluor 488-conjugated anti-human IgG (H+L) (invitrogen, A-11013) at working concentration was added, and it was incubated in the dark at 4°C for 40 minutes. After centrifugal washing 3 times with FACS buffer at 300 g, the geometric mean fluorescence intensity was detected on a BD FACS CantoII flow cytometer, and the binding EC50 value of the Claudin18.2 antibody to NUGC4 cells stably expressing Claudin18.2 was calculated. The results are shown in Figure 1 .

[0488] Test Example 3: Antibody Endocytosis Experiment

[0489] The test Claudin18.2 antibody pre-labeled with DyLight 488 NHS Ester (thermofisher, 46403) was added to 1×10 6 / ml NUGC4 cells stably expressing Claudin18.2 at a final concentration of 5 μg / ml, incubated on ice in the dark for 1 hour, centrifugally washed 3 times with pre-cooled FACS buffer (pH 7.4 PBS, 2% fetal bovine serum), the supernatant was removed, pre-warmed complete medium was added, and it was placed in a 37°C 5% CO 2 cell incubator. Cells were taken out at 0, 0.5, 1, 2, and 4 hours respectively, placed on ice and stored in the dark. After all samples were collected, the supernatant was removed by low-temperature centrifugation at 300 g, elution buffer (pH 1.7 0.05 M glycine, 0.1 M sodium chloride) was added, incubated at room temperature for 7 minutes, centrifugally washed 1 time with FACS buffer at 300 g, and the geometric mean fluorescence intensity was detected on a BD FACSCantoII flow cytometer, and the endocytosis efficiency of the Claudin18.2 antibody to NUGC4 cells stably expressing Claudin18.2 was calculated. The results showed (see Figure 2 ) that the humanized antibody had good endocytosis efficiency.

[0490] Test Example 4: Determination of Antibody Affinity Based on Flow Cytometry

[0491] On the day of the experiment, HEK293 / hClaudin18.2 cells were collected into a U-bottom 96-well plate, 1×10 5 to 2×10 5cells. Add Claudin18.2 antibody with an initial concentration of 5 μg / ml and 2-fold serial dilution (12 concentration points), incubate at 4 °C for 1 hour. The positive control is IMAB362, and a negative control without antibody is also set. Centrifuge to remove the antibody, then add 100 μl / well of FITC anti-human IgG Fc antibody (200×), incubate at 4 °C in the dark for 30 minutes, wash twice with PBS + 2% FBS, and prepare for flow cytometry. Start BD FACS CantoII, after preheating, open BD FACSDiva software, create a new experiment, detect the HEK293 / hClaudin18.2 negative control sample, adjust the FSC and SSC voltages to appropriate values and save. According to the Quantum TM FITC-5MESF Kit instructions, detect blank sample B and standard curve 1 respectively, adjust the FITC voltage to an appropriate value and save. Detect the samples in the U-bottom 96-well plate at the saved voltages and record the data. Use Flowjo software to analyze the experimental data to obtain the Geo Mean value, and fit the MESF-Geo Mean standard curve according to the Quantum TM FITC-5MESF Kit instructions, calculate the molar concentration and free antibody concentration of the Claudin18.2 antibody bound to HEK293 / hClaudin18.2 cells based on the fluorescence value of the FITC anti-human IgG Fc antibody concentration, and calculate the Bmax and dissociation constant KD of the antibody using the Scatchard plot method. The results are shown in Table 14.

[0492] Table 14. Affinity of humanized antibody at the cellular level

[0493] antibody IMAB362 h1901-11 h1902-5 KD (nM) 10.2 6.8 1.64

[0494] Test Example 5: Evaluation of the ADCC effect of the antibody

[0495] Digest various NUGC4 cells (high, medium, and low expression of Claudin18.2), centrifuge at 1000 rpm, and resuspend and count. Resuspend the cells at a density of 3×10 5 cells / ml in phenol red-free RPMI 1640 supplemented with 10% FBS (New Zealand ultra-low IgG fetal bovine serum, Gibco, 1921005PJ) (Gibco, 11835-030). In a 96-well plate (Corning, 3903), add 25 μl of cells (7500 cells / well) to each well. Dilute the antibody in the above phenol red-free medium to prepare a 3-fold antibody dilution, and add 25 μl / well of the antibody to the cell plate. Incubate in an incubator at 37 °C and 5% CO 2 for 0.5 hours.

[0496] Collect effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells), centrifuge at 1000 rpm, and resuspend and count. Resuspend the cells at a density of 3×10 6 cells / ml in phenol red-free RPMI 1640 supplemented with 10% FBS (New Zealand ultra-low IgG fetal bovine serum), and add 25 μl of cells (7.5×10 4 cells / well) to each well of the experimental plate. Incubate in an incubator at 37 °C and 5% CO 2 for 6 hours.

[0497] Add 75 μl / well of Bright-Glo (Promega, E2610) to each well of the experimental plate, and detect the chemiluminescence using a microplate reader (PerkinElmer, VITOR3).

[0498] The results showed (see Table 15 and Figures 3A - 3C ), in NUGC4 cells with low ( Figure 3A )-medium ( Figure 3B )-high ( Figure 3C ) levels of Claudin18.2 expression, both antibodies h1901-11 and h1902-5 showed strong ADCC activity.

[0499] Table 15. ADCC effector unit IC50 (ng / ml) of antibodies in NUGC4 cells with different expression levels of Claudin18.2

[0500] Claudin18.2 expression level h1901-11 h1902-5 IMAB362 Low expression 22.42 35.46 183.4 Medium expression 15.35 30.00 210.4 High expression 26.17 32.16 132.6

[0501] Test Example 6: Cell Activity Experiment of ADC Molecule

[0502] In this experiment, the killing effect of the ADC molecule on human gastric cancer cell lines was detected in vitro using the CellTiter-Glo Luminescence Cell Viability Assay. On the first day, collect NUGC4-claudin18.2 low-expression, NUGC4-claudin18.2 medium-expression, and NUGC4-claudin18.2 high-expression cells, adjust the density to 2.5×10 4 / ml, add 90 μl / well to a 96-well white transparent bottom plate, approximately 2500 cells per well. Incubate overnight in an incubator at 37 °C and 5% CO 2 . On the second day, dilute the samples in a U-bottom 96-well plate, starting concentration is 5 μM, 4× serial dilution, 9 concentration points, and add 10 μl / well of the diluted samples to the cell plate. Incubate at 37 °C and 5% CO 2Cultivate for 6 days. On the eighth day, take out the cell culture plate, add 50 μl / well CellTiter-Glo Reagent, place it at room temperature for 2 to 3 minutes, and read the luminescence value on the PHERAstar FS microplate reader. Use GraphPad Prism software for data analysis. See Table 16.

[0503] Table 16. In vitro cell killing experiment of ADC

[0504]

[0505] Test Example 7: In vivo pharmacodynamic evaluation of ADC molecule

[0506] Subcutaneously inoculate human gastric cancer cell NUGC4 (Claudin18.2 moderately expressed) cells (5×10 6 containing 50% matrigel matrix gel / per mouse) in the right rib subcutaneous of Balb / c nude mice. Group on day 0, 8 mice / group, a total of 8 groups. The average tumor volume is about 84.41 mm 3 .

[0507] Inject ADC intraperitoneally, a total of 3 doses, inject 10 g / 0.1 ml per mouse according to body weight, and administer the drug on day 0, 4, and 11 respectively.

[0508] Inject ADC intraperitoneally on the day of grouping, a total of 4 doses, administer the drug at intervals of 5 days, and inject 10 g / 0.1 ml per mouse according to body weight.

[0509] Measure the tumor volume and body weight 2 times a week, and record the data.

[0510] Use Excel 2003 statistical software: calculate the average value as avg; calculate the SD value as STDEV; calculate the SEM value as STDEV / SQRT; calculate the P value of the difference between groups as TTEST.

[0511] The formula for calculating the tumor volume (V) is: V = 1 / 2 × L 长 × L 短 2

[0512] Relative volume (RTV) = VT / V0

[0513] Tumor inhibition rate (%) = (CRTV - TRTV) / CRTV (%)

[0514] Where V0 and VT are the tumor volumes at the start of the experiment (the first day of drug administration is day 0) and at the end of the experiment, respectively. CRTV and TRTV are the relative tumor volumes of the blank control group (Vehicle) and the experimental group at the end of the experiment. The results are shown in Table 17 and Figure 4, Figure 5 .

[0515] Table 17. Results of the anti-tumor experiment of ADC

[0516]

[0517] vs blank control group: **p < 0.01.

[0518] II. Preparation

[0519] The equipment used in the preparation and detection of the preparation and the result calculation method are as follows:

[0520] SEC size exclusion chromatography:

[0521] An analytical method for separating solutes based on the relative relationship between the pore size of the gel pores and the coil size of the polymer sample molecules.

[0522] SEC% (percentage of SEC monomer content) = A monomer / A total * 100% (A monomer is the peak area of the main peak monomer in the sample, and A total is the sum of all peak areas).

[0523] Instruments for SEC determination: Agilent 1260; Column: waters, XBrige SEC (300×7.8mm 3.5μm).

[0524] CE capillary gel electrophoresis:

[0525] An electrophoresis method in which the gel is transferred into the capillary as the supporting medium and separated according to the molecular weight of the sample under a certain voltage.

[0526] Reduced CE purity percentage = A main peak / A total * 100% (A main peak is the peak area of the light chain main peak + heavy chain main peak in the sample, and A total is the sum of all peak areas.

[0527] Instruments for CE determination: Beckman model plus800.

[0528] Osmotic pressure determination:

[0529] The osmotic pressure is determined by the freezing point method. Based on the proportional relationship between the freezing point depression value and the molar concentration of the solution, a highly sensitive temperature sensing element is used to measure the freezing point of the solution, and the electric quantity is converted into the osmotic pressure. Instrument manufacturer: Loser, model OM815.

[0530] Protein concentration determination:

[0531] Since the drug in the antibody-drug conjugate has absorption at 280 nm, the protein concentration is corrected using the following formula,

[0532] A280 = Cd * ε280d + Cmab * ε280mab;

[0533] A370 = Cd * ε370d;

[0534] Cd represents the concentration of the drug, Cmab represents the concentration of the protein, ε280d represents the extinction coefficient of the drug at 280 nm, ε280mab represents the extinction coefficient of the protein at 280 nm, and ε370d represents the extinction coefficient of the drug at 370 nm. ε280mab = 1.49 mg-1 * cm-1 * ml, ε280d = 5000 (molar extinction coefficient of the drug at 280 nm) / 1074.13 (molecular weight of the drug) = 4.65 mg-1 * cm-1 * mL, ε370d = 19000 (molar extinction coefficient of the drug at 370 nm) / 1074.13 (molecular weight of the drug) = 17.69 mg-1 * cm-1 * mL. The above extinction coefficients are mass extinction coefficients.

[0535] Protein concentration measuring instrument: UV-visible spectrophotometer, model: Nano Drop oneC, with an optical path of 1 mm.

[0536] Example 2-1: Screening of formulation buffer systems and pH values

[0537] Prepare formulations containing 20 mg / mL (protein concentration) of ADC-9 and the following different buffer systems, as well as 0.1 mg / mL polysorbate 80 (PS80).

[0538] 1) 10 mM citric acid - sodium citrate (CA), pH 5.5

[0539] 2) 10 mM succinic acid - sodium succinate (SA), pH 5.0

[0540] 3) 10 mM succinic acid - sodium succinate, pH 5.5

[0541] 4) 10 mM histidine hydrochloride (His-HCl), pH 5.5

[0542] 5) 10 mM histidine hydrochloride, pH 6.0

[0543] 6) 10 mM histidine hydrochloride, pH 6.5

[0544] 7) 10 mM histidine - acetate (His-AA), pH 5.0

[0545] 8) 10 mM histidine - acetate, pH 5.5

[0546] 9) 10 mM phosphate buffer (PB), pH 6.5.

[0547] Filter, fill, stopper, and crimp each preparation. Take samples for high-temperature stability (40 °C) and shaking (25 °C, 300 rpm) studies, and examine appearance, SEC, and reduced CE. The results are shown in Table 18.

[0548] After shaking for 11 days, only the samples of No. 2), 3), 7), and 9) were clear in appearance; after standing at 40 °C for 15 days, the samples of No. 1), 2), 3), 7), and 8) were clear in appearance. That is, from the perspective of appearance, the preparations of the samples of No. 2), 3), and 7) were superior.

[0549] After standing at 40 °C for 15 days, the samples were detected by SEC. The results showed that the monomer reduction of the samples of No. 4), 5), 6), 7), and 8) was about 4%; the monomer reduction of other preparations was 7%-10%.

[0550] After standing at 40 °C for 15 days, the samples were detected by reduced CE. The results showed that the peak of the samples of No. 4), 5), 7), and 8) decreased by about 1%-2%, which was superior to other samples.

[0551] Based on the above data, the sample of No. 7), namely the 10 mM His-AA, pH 5.0 preparation, was superior to the other preparations in terms of appearance and various chemical detection items. Therefore, 10 mM His-AA, pH 5.0 was selected as the final buffer.

[0552] Table 18. pH and buffer stability results

[0553]

[0554]

[0555] Note: "D" in the table represents days. For example, D3 represents 3 days, and so on; D0 represents the start of the experiment, and the same applies hereinafter.

[0556] Example 2-2: Screening of surfactant type and concentration

[0557] Prepare preparations containing different types and concentrations of polysorbate, and containing 10 mM His-AA, pH 5.0 buffer, 80 mg / mL sucrose, and ADC-9 with a protein concentration of 20 mg / mL. Filter, fill, stopper, and crimp each preparation. Conduct high-temperature stability studies (40 °C) and freeze-thaw studies on the samples. The freeze-thaw study is to place at room temperature for three days (25 °C D3) after 5 freeze-thaw cycles (FT5C, 35 °C -2 to 8 °C), and examine appearance, SEC, and reduced CE. The specific preparation design is shown in Table 19.

[0558] The results are shown in Table 20. The experimental results show that the preparation with 0.2 mg / ml PS80 is the best in terms of appearance and chemical detection under various conditions.

[0559] Based on the above results, the type and concentration of the surfactant are determined to be 0.2 mg / ml PS80.

[0560] Table 19. Screening of the types and concentrations of polysorbates in the preparation

[0561]

[0562] Note: PS20 represents polysorbate 20.

[0563] Table 20. Screening results of the types of polysorbates

[0564]

[0565]

[0566] Remarks: In the table, "M" represents month, M1 represents 1 month, and so on. The same applies hereinafter.

[0567] Example 2 - 3: Screening of sugar types

[0568] Prepare preparations containing sucrose, trehalose, and mannitol respectively, which also contain 10 mM His-AA (pH 5.0) buffer, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) of ADC-9. Filter, fill, stopper, and crimp the cap for each preparation. Conduct high-temperature stability studies (40 °C), -35 °C / 4 °C freeze-thaw cycles, and place the samples at room temperature for 3 days to investigate appearance, SEC, and reduced CE.

[0569] The results are shown in Table 21. Under the freeze-thaw conditions of preparations with different sugar types, the appearance of the samples with sucrose is better than those with trehalose or mannitol. The SEC test results show that the samples with sucrose or trehalose are better than those with mannitol; after being placed at 40 °C for one month, the appearance of the samples with sucrose is better than those with trehalose or mannitol, and the SEC and reduced CE test results also show that the samples with sucrose are slightly better than those with trehalose.

[0570] Table 21. Screening results of sugar type solutions

[0571]

[0572] Prepare preparations containing sucrose, trehalose, and mannitol respectively, which also contain 10 mM His-AA (pH 5.0) buffer, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) ADC-9. Filter, fill, semi-cork, freeze-dry, cork, and crimp each preparation, and place it in a high-temperature stability study (40 °C) to examine appearance, SEC, and reduced CE. The freeze-drying process refers to the freeze-drying process parameters 1 in Table 22.

[0573] Table 22. Freeze-drying process parameters 1

[0574]

[0575]

[0576] After placing at 40 °C for one month, the detection results of SEC (see Table 23) show that the samples using sucrose are slightly better than those using trehalose, and much better than those using mannitol. The detection results of reduced CE (see Table 23) show that the samples using sucrose and those using trehalose are comparable, and both are better than those using mannitol.

[0577] Table 23. Screening results of sugar-based lyophilizers

[0578]

[0579] Examples 2-4: Appearance optimization experiment of freeze-dried samples

[0580] Prepare the stock solution according to 10 mM His-AA, pH 5.0, 80 mg / mL sucrose, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) ADC-9. After filtration and filling, freeze-dry according to the freeze-drying process parameters 1 (see Table 22), and examine the appearance of the freeze-dried samples. The freeze-dried samples are flat white powder cakes on the surface, but the bottom edges of the powder cakes are slightly shrunk.

[0581] Further lower the sugar concentration of the samples to 60 mg / mL. Prepare the stock solution according to 10 mM His-AA, pH 5.0, 60 mg / mL sucrose, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) ADC-9. After filtration and filling, freeze-dry according to the freeze-drying process parameters 1 in Table 22. After freeze-drying, the surface of the powder cake is flat without shrinkage, and the bottom edges of the powder cake are slightly shrunk at the bottom.

[0582] The sugar concentration was reduced to 40 mg / ml. However, the osmotic pressure of the final product was too low at this time, and there was a risk of low osmotic pressure during clinical administration. To ensure the osmotic pressure of the final product, the ionic strength of the buffer was increased to 30 mM. The stock solution was prepared according to 30 mM His-AA, pH 5.0, 40 mg / mL sucrose, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) ADC-9. After filtration and filling, it was lyophilized according to the lyophilization process parameters 1. After lyophilization, the surface of the sample powder cake was flat without collapse, and the bottom edge of the powder cake was intact.

[0583] The results of the three preparations are shown in Table 24.

[0584] Table 24. Appearance of the samples after lyophilization

[0585]

[0586]

[0587] Example 2-5: Stability of the samples after lyophilization

[0588] The stock solution was prepared according to the preparations in Table 25. After filtration and filling, it was lyophilized according to the lyophilization process parameters 1 (see Table 22). The samples after lyophilization were placed at 40°C under condition M1 and then reconstituted to detect their stability changes.

[0589] The stability results are shown in Table 25. The reduced CE of the lyophilized samples of formulation 3 decreased by about 3.7% under the condition of 40°C M1. There were no obvious changes in the chemical detection items of the other two formulations, and their stability was significantly better than that of formulation 3. The pH of the stock solution of formulation 2 before lyophilization was 5.04, and the pH of the reconstituted solution after lyophilization was 5.27.

[0590] Table 25. Results of formulation stability

[0591]

[0592] Example 2-6: Stability of the lyophilized preparation solution

[0593] The preparation was prepared according to the preparations in Table 26. After filtration, filling, stoppering, and capping, it was subjected to freeze-thaw cycles at -35°C / 4°C and stored at room temperature for 3 days, shaken for 11 days, and studied for high-temperature stability (40°C). The appearance, SEC, and reduced CE changes of the samples were investigated under the corresponding conditions.

[0594] The stability results are shown in Table 26. After reducing the sucrose concentration and increasing the ionic strength of the buffer, there were no obvious differences in the appearance and purity changes between formulation 2 and formulation 1. The reduction of CE of formulation 2 was slightly better than that of formulation 1 under high-temperature conditions.

[0595] Table 26. Results of formulation stability

[0596] Sequence Listing <110> Jiangsu Hengrui Medicine Co., Ltd. Shanghai Hengrui Medicine Co., Ltd. <120> A Pharmaceutical Composition Containing an Antibody-Drug Conjugate and Its Use <130> 721106CPCT <140> PCT / CN2021 / <141> 2021-09-30 <150> 202011061863.1 <151> 2020-09-30 <150> 202111069020.0 <151> 2021-09-13 <160> 55 <170> SIPOSequenceListing 1.0 <210> 1 <211> 261 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <223> Claudin18.2 protein <400> 1 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 2 <211> 3350 <212> DNA <213> Homo sapiens <220> <221> gene <223> Claudin18.2 <400> 2 agaattgcgc tgtccacttg tcgtgtggct ctgtgtcgac actgtgcgcc accatggccg 60 tgactgcctg tcagggcttg gggttcgtgg tttcactgat tgggattgcg ggcatcattg 120 ctgccacctg catggaccag tggagcaccc aagacttgta caacaacccc gtaacagctg 180 ttttcaacta ccaggggctg tggcgctcct gtgtccgaga gagctctggc ttcaccgagt 240 gccggggcta cttcaccctg ctggggctgc cagccatgct gcaggcagtg cgagccctga 300 tgatcgtagg catcgtcctg ggtgccattg gcctcctggt atccatcttt gccctgaaat 360 gcatccgcat tggcagcatg gaggactctg ccaaagccaa catgacactg acctccggga 420 tcatgttcat tgtctcaggt ctttgtgcaa ttgctggagt gtctgtgttt gccaacatgc 480 tcatgttcat tgtctcaggt ctttgtgcaa ttgctggagt gtctgtgttt gccaacatgc 480 tggtgactaa cttctggatg tccacagcta acatgtacac cggcatgggt gggatggtgc 540 tggtgactaa cttctggatg tccacagcta acatgtacac cggcatgggt gggatggtgc 540 agactgttca gaccaggtac acatttggtg cggctctgtt cgtgggctgg gtcgctggag 600 agactgttca gaccaggtac acatttggtg cggctctgtt cgtgggctgg gtcgctggag 600 gcctcacact aattgggggt gtgatgatgt gcatcgcctg ccggggcctg gcaccagaag 660 gcctcacact aattgggggt gtgatgatgt gcatcgcctg ccggggcctg gcaccagaag 660 aaaccaacta caaagccgtt tcttatcatg cctcaggcca cagtgttgcc tacaagcctg 720 aaaccaacta caaagccgtt tcttatcatg cctcaggcca cagtgttgcc tacaagcctg 720 gaggcttcaa ggccagcact ggctttgggt ccaacaccaa aaacaagaag atatacgatg 780 gaggcttcaa ggccagcact ggctttgggt ccaacaccaa aaacaagaag atatacgatg 780 gaggtgcccg cacagaggac gaggtacaat cttatccttc caagcacgac tatgtgtaat 840 gaggtgcccg cacagaggac gaggtacaat cttatccttc caagcacgac tatgtgtaat 840 gctctaagac ctctcagcac gggcggaaga aactcccgga gagctcaccc aaaaaacaag 900 gctctaagac ctctcagcac gggcggaaga aactcccgga gagctcaccc aaaaaacaag 900 gagatcccat ctagatttct tcttgctttt gactcacagc tggaagttag aaaagcctcg 960 gagatcccat ctagatttct tcttgctttt gactcacagc tggaagttag aaaagcctcg 960 atttcatctt tggagaggcc aaatggtctt agcctcagtc tctgtctcta aatattccac 1020 atttcatctt tggagaggcc aaatggtctt agcctcagtc tctgtctcta aatattccac 1020 cataaaacag ctgagttatt tatgaattag aggctatagc tcacattttc aatcctctat 1080 cataaaacag ctgagttatt tatgaattag aggctatagc tcacattttc aatcctctat 1080 ttcttttttt aaatataact ttctactctg atgagagaat gtggttttaa tctctctctc 1140 ttcttttttt aaatataact ttctactctg atgagagaat gtggttttaa tctctctctc 1140 acattttgat gatttagaca gactccccct cttcctccta gtcaataaac ccattgatga 1200 tctatttccc agcttatccc caagaaaact tttgaaagga aagagtagac ccaaagatgt 1260 tattttctgc tgtttgaatt ttgtctcccc acccccaact tggctagtaa taaacactta 1320 ctgaagaaga agcaataaga gaaagatatt tgtaatctct ccagcccatg atctcggttt 1380 tcttacactg tgatcttaaa agttaccaaa ccaaagtcat tttcagtttg aggcaaccaa 1440 acctttctac tgctgttgac atcttcttat tacagcaaca ccattctagg agtttcctga 1500 gctctccact ggagtcctct ttctgtcgcg ggtcagaaat tgtccctaga tgaatgagaa 1560 aattattttt tttaatttaa gtcctaaata tagttaaaat aaataatgtt ttagtaaaat 1620 gatacactat ctctgtgaaa tagcctcacc cctacatgtg gatagaagga aatgaaaaaa 1680 taattgcttt gacattgtct atatggtact ttgtaaagtc atgcttaagt acaaattcca 1740 tgaaaagctc actgatccta attctttccc tttgaggtct ctatggctct gattgtacat 1800 gatagtaagt gtaagccatg taaaaagtaa ataatgtctg ggcacagtgg ctcacgcctg 1860 taatcctagc actttgggag gctgaggagg aaggatcact tgagcccaga agttcgagac 1920 tagcctgggc aacatggaga agccctgtct ctacaaaata cagagagaaa aaatcagcca 1980 gtcatggtgg cctacacctg tagtcccagc attccgggag gctgaggtgg gaggatcact 2040 tgagcccagg gaggttgggg ctgcagtgag ccatgatcac accactgcac tccagccagg 2100 tgacatagcg agatcctgtc taaaaaaata aaaaataaat aatggaacac agcaagtcct 2160 aggaagtagg ttaaaactaa ttctttaaaa aaaaaaaaaa gttgagcctg aattaaatgt 2220 aatgtttcca agtgacaggt atccacattt gcatggttac aagccactgc cagttagcag 2280 tagcactttc ctggcactgt ggtcggtttt gttttgtttt gctttgttta gagacggggt 2340 ctcactttcc aggctggcct caaactcctg cactcaagca attcttctac cctggcctcc 2400 caagtagctg gaattacagg tgtgcgccat cacaactagc tggtggtcag ttttgttact 2460 ctgagagctg ttcacttctc tgaattcacc tagagtggtt ggaccatcag atgtttgggc 2520 aaaactgaaa gctctttgca accacacacc ttccctgagc ttacatcact gcccttttga 2580 gcagaaagtc taaattcctt ccaagacagt agaattccat cccagtacca aagccagata 2640 ggccccctag gaaactgagg taagagcagt ctctaaaaac tacccacagc agcattggtg 2700 caggggaact tggccattag gttattattt gagaggaaag tcctcacatc aatagtacat 2760 atgaaagtga cctccaaggg gattggtgaa tactcataag gatcttcagg ctgaacagac 2820 tatgtctggg gaaagaacgg attatgcccc attaaataac aagttgtgtt caagagtcag 2880 agcagtgagc tcagaggccc ttctcactga gacagcaaca tttaaaccaa accagaggaa 2940 gtatttgtgg aactcactgc ctcagtttgg gtaaaggatg agcagacaag tcaactaaag 3000 aaaaaagaaa agcaaggagg agggttgagc aatctagagc atggagtttg ttaagtgctc 3060 tctggatttg agttgaagag catccatttg agttgaaggc cacagggcac aatgagctct 3120 cccttctacc accagaaagt ccctggtcag gtctcaggta gtgcggtgtg gctcagctgg 3180 gtttttaatt agcgcattct ctatccaaca tttaattgtt tgaaagcctc catatagtta 3240 gattgtgctt tgtaattttg ttgttgttgc tctatcttat tgtatatgca ttgagtatta 3300 acctgaatgt tttgttactt aaatattaaa aacactgtta tcctacagtt 3350 <210> 3 <211> 120 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> mAb1901 mouse-derived heavy chain variable region <400> 3 Glu Val Gln Leu Met Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Glu Met Gly Leu Glu Trp Ile 35 40 45 Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 4 <211> 113 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> mAb1901 mouse-derived antibody light chain variable region <400> 4 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr His Cys Gln Asn 85 90 95 Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 5 <211> 118 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> Variable region of the heavy chain of mAb1902 murine antibody <400> 5 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Pro Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 6 <211> 113 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> Light chain variable region of mAb1902 murine antibody <400> 6 Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 7 <211> 330 <212> PRT <213> Homo sapiens <220> <221> DOMAIN <223> Heavy chain constant region of human IgG1 antibody <400> 7 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 8 <211> 107 <212> PRT <213> Homo sapiens <220> <221> DOMAIN <223> Human antibody kappa light chain constant region <400> 8 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> mAb1901 HCDR1 <400> 9 Asp Tyr Gly Ile His 1 5 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> mAb1901 HCDR2 <400> 10 Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val Lys 1 5 10 15 Gly <210> 11 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> mAb1901 HCDR3 <400> 11 Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr 1 5 10 <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> mAb1901 LCDR1 <400> 12 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> mAb1901 LCDR2 <400> 13 Gly Ala Ser Thr Arg Ala Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> mAb1901 LCDR3 <400> 14 Gln Asn Asp Leu Tyr Tyr Pro Leu Thr 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> mAb1902 HCDR1 <400> 15 Ser Tyr Trp Met His 1 5 <210> 16 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> mAb1902 HCDR2 <400> 16 Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> mAb1902 HCDR3 <400> 17 Leu Lys Thr Gly Asn Ser Phe Asp Tyr 1 5 <210> 18 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> mAb1902 LCDR1 <400> 18 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> mAb1902 LCDR2 <400> 19 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> mAb1902 LCDR3 <400> 20 Gln Asn Ala Tyr Thr Tyr Pro Phe Thr 1 5 <210> 21 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VL1 <400> 21 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 22 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VL2 <400> 22 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 23 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VL3 <400> 23 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Ile Tyr His Cys Gln Asn 85 90 95 Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 24 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VH1 <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 25 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VH2 <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 26 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VH3 <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 27 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VH4 <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 28 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VL11 <400> 28 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 29 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VL12 <400> 29 Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 30 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VL13 <400> 30 Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 31 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VH11 <400> 31 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 32 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VH12 <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 33 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VH13 <400> 33 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 34 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> VH14 <400> 34 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 35 <211> 450 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> ch1901 heavy chain <400> 35 Glu Val Gln Leu Met Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Glu Met Gly Leu Glu Trp Ile 35 40 45 Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 36 <211> 220 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> ch1901 light chain <400> 36 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr His Cys Gln Asn 85 90 95 Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 37 <211> 448 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> ch1902 heavy chain <400> 37 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Pro Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 38 <211> 220 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> ch1902 light chain <400> 38 Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 39 <211> 220 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> L1 <400> 39 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 40 <211> 220 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> L2 <400> 40 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 41 <211> 220 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> L3 <400> 41 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Ile Tyr His Cys Gln Asn 85 90 95 Asp Leu Tyr Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 42 <211> 450 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> H1 <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 43 <211> 450 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> H2 <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 44 <211> 450 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> H3 <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 45 <211> 450 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> H4 <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Arg Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Met Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Thr Arg Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 46 <211> 220 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> L11 <400> 46 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 47 <211> 220 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> L12 <400> 47 Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 48 <211> 220 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> L13 <400> 48 Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 49 <211> 448 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> H11 <400> 49 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 50 <211> 448 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> H12 <400> 50 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 51 <211> 448 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> H13 <400> 51 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 52 <211> 448 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> H14 <400> 52 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Leu Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Lys Thr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 53 <211> 448 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> IMAB-362 heavy chain <400> 53 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 54 <211> 220 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> IMAB-362 light chain <400> 54 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 55 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Tetrapeptide linker <400> 55 Gly Gly Phe Gly 1 4

Claims

1. A pharmaceutical composition comprising an anti-Claudin18.2 antibody-drug conjugate and a buffer, wherein the anti-Claudin18.2 antibody in the anti-Claudin18.2 antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein: the buffer is a histidine salt buffer, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 respectively.

2. The pharmaceutical composition according to claim 1, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is as shown in SEQ ID NO: 31 and the light chain variable region is as shown in SEQ ID NO:

29.

3. The pharmaceutical composition according to claim 1, wherein the anti-Claudin18.2 antibody comprises a heavy chain as shown in SEQ ID NO: 49 and a light chain as shown in SEQ ID NO:

47.

4. The pharmaceutical composition according to claim 1, wherein the anti-Claudin18.2 antibody-drug conjugate has a structure represented by the general formula (Pc-L-Y-D): wherein: Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b are the same or different and each independently selected from a hydrogen atom, a deuterium atom, a halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 deuterated alkyl, C 1-6 alkoxy, hydroxy, amino, cyano, nitro, C 1-6 hydroxyalkyl and C 3-7 cycloalkyl; Alternatively, R a and R b together with the carbon atom to which it is attached form a C 3-7 cycloalkyl group; R 1 Selected from halogen, C 1-6 haloalkyl, C 1-6 deuterated alkyl, C 3-7 cycloalkyl, C 3-7 cycloalkyl C 1-6 alkyl and C 1-6 alkoxy C 1-6 alkyl; R 2 selected from a hydrogen atom, a halogen, C 1-6 haloalkyl, C 1-6 deuterated alkyl, C 3-7 cycloalkyl, C 3-7 cycloalkyl C 1-6 alkyl and C 1-6 alkoxy C 1-6 alkyl; Alternatively, R 1 and R 2 together with the carbon atom to which it is attached form a C 3-7 cycloalkyl group; Alternatively, R a and R 2 together with the carbon atom to which it is attached form a C 3-7 cycloalkyl group; m is an integer from 0 to 4; n is from 1 to 10, n is a decimal or an integer; L is a linker unit; Pc is an anti-Claudin18.2 antibody.

5. The pharmaceutical composition according to claim 1, wherein the anti-Claudin18.2 antibody-drug conjugate has a structure represented by the following formula: wherein: n is from 2 to 8, n is a decimal or an integer; Pc is an anti-Claudin18.2 antibody.

6. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a surfactant.

7. The pharmaceutical composition according to claim 6, wherein the surfactant is polysorbate.

8. The pharmaceutical composition according to claim 6, wherein the surfactant is polysorbate 80 or polysorbate 20.

9. The pharmaceutical composition according to claim 6, wherein the surfactant is polysorbate 80.

10. The pharmaceutical composition according to claim 6, wherein the surfactant concentration is from 0.05 mg / mL to 0.5 mg / mL.

11. The pharmaceutical composition according to claim 6, wherein the surfactant concentration is from 0.1 mg / mL to 0.2 mg / mL.

12. The pharmaceutical composition according to claim 6, wherein the surfactant concentration is 0.2 mg / mL.

13. The pharmaceutical composition according to claim 1, wherein the composition further comprises a sugar.

14. The pharmaceutical composition according to claim 13, wherein the sugar is selected from sucrose, mannitol and trehalose.

15. The pharmaceutical composition according to claim 13, wherein the sugar is sucrose.

16. The pharmaceutical composition according to claim 13, wherein the sugar concentration is 20 mg / mL to 100 mg / mL.

17. The pharmaceutical composition according to claim 13, wherein the sugar concentration is 40 mg / mL to 80 mg / mL.

18. The pharmaceutical composition according to claim 13, wherein the sugar concentration is 40 mg / mL.

19. The pharmaceutical composition according to claim 1, wherein the concentration of the anti-Claudin 18.2 antibody-drug conjugate is 1 mg / mL to 100 mg / mL in terms of protein concentration.

20. The pharmaceutical composition according to claim 19, wherein the concentration of the anti-Claudin 18.2 antibody-drug conjugate is 10 mg / mL to 30 mg / mL in terms of protein concentration.

21. The pharmaceutical composition according to claim 19, wherein the concentration of the anti-Claudin 18.2 antibody-drug conjugate is 20 mg / mL in terms of protein concentration.

22. The pharmaceutical composition according to claim 1, wherein the buffer is histidine-acetate buffer.

23. The pharmaceutical composition according to claim 1, wherein the concentration of the buffer is 5 mM to 50 mM.

24. The pharmaceutical composition according to claim 23, wherein the concentration of the buffer is 10 mM to 30 mM.

25. The pharmaceutical composition according to claim 23, wherein the concentration of the buffer is 30 mM.

26. The pharmaceutical composition according to claim 1, wherein the pH of the pharmaceutical composition is 5.0 - 6.

5.

27. The pharmaceutical composition according to claim 26, wherein the pH of the pharmaceutical composition is 5.0 - 5.

5.

28. The pharmaceutical composition according to claim 26, wherein the pH of the pharmaceutical composition is 5.0 - 5.

3.

29. The pharmaceutical composition according to claim 1, which comprises the following components: (a) The anti-Claudin 18.2 antibody-drug conjugate at a concentration of 10 mg / mL to 30 mg / mL in terms of protein concentration, (b) polysorbate at a concentration of 0.1 mg / mL to 0.2 mg / mL, (c) sugar at a concentration of 40 mg / mL to 80 mg / mL, and (d) histidine buffer at a concentration of 10 mM to 30 mM; the pH of the pharmaceutical composition is 5.0 - 5.

5.

30. The pharmaceutical composition according to claim 1, wherein the pH of the pharmaceutical composition comprises the following components: (a) The anti-Claudin 18.2 antibody-drug conjugate at a concentration of 20 mg / mL in terms of protein concentration, (b) polysorbate 80 at a concentration of 0.2 mg / mL, (c) sucrose at a concentration of 40 mg / mL, and (d) 30 mM histidine-acetate buffer, the pH of the pharmaceutical composition is 5.0 - 5.

3.

31. A pharmaceutical composition, which comprises: An anti-Claudin 18.2 antibody-drug conjugate at a concentration of 20 mg / mL in terms of protein concentration Polysorbate 80 at 0.2 mg / mL, sucrose at 40 mg / mL, and 30 mM histidine-acetate buffer; The pH of the pharmaceutical composition is 5.0 to 5.3; The anti-Claudin18.2 antibody-drug conjugate has the structure shown in the following formula: Wherein: n is from 2 to 8, and n is a decimal or an integer; Pc is an anti-Claudin18.2 antibody, which comprises a heavy chain as shown in SEQ ID NO: 49 and a light chain as shown in SEQ ID NO:

47.

32. A freeze-dried preparation containing an antibody-drug conjugate, characterized in that the preparation forms the pharmaceutical composition according to any one of claims 1 to 31 after reconstitution.

33. A method for preparing a freeze-dried preparation containing an antibody-drug conjugate, which includes the step of freeze-drying the pharmaceutical composition according to any one of claims 1 to 31.

34. A freeze-dried preparation containing an antibody-drug conjugate, which is obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 31.

35. A reconstituted solution containing an antibody-drug conjugate, characterized in that the reconstituted solution is prepared by reconstituting the freeze-dried preparation according to claim 32 or 34.

36. The reconstituted solution according to claim 35, wherein the reconstituted solution contains the following components: (a) The anti-Claudin18.2 antibody-drug conjugate at a protein concentration of 20 mg / mL, (b) Polysorbate 80 at 0.2 mg / mL, (c) Sucrose at 40 mg / mL, and (d) 30 mM histidine-acetate buffer, and the pH of the reconstituted solution is 5.0 - 5.

3.

37. An article, which includes a container containing the pharmaceutical composition according to any one of claims 1 to 31, the freeze-dried preparation according to claim 32 or 34, or the reconstituted solution according to claim 35.

38. Use of the pharmaceutical composition according to any one of claims 1 to 31, or the freeze-dried preparation according to claim 32 or 34, or the reconstituted solution according to claim 35, or the article according to claim 37 in the preparation of a medicament for treating gastric cancer.

Images